MODULATION OF pre-mRNA USING SPLICE MODULATING OLIGONUCLEOTIDES AS THERAPEUTIC AGENTS IN THE TREATMENT OF DISEASE

ABSTRACT

The present invention encompasses a class of compounds known as splice modulating oligonucleotides (SMOs) that modulate pre-mRNA splicing, thereby affecting expression and functionality of a specific protein in a cell. The present invention further provides compositions and methods for modulating pre-mRNA splicing using a SMO of the invention to abrogate disease-causing mutations in a protein. Accordingly, the present invention provides compositions and methods of treating a subject at risk of, susceptible to, or having a disease, disorder, or condition associated with aberrant or unwanted target pre-mRNA expression or activity.

BACKGROUND OF THE INVENTION

Approximately 90,000 known human proteins are the product of about20,000 human genes. It is estimated that roughly 75% of human genes aresubject to alternative splicing. Alternative splicing is the processresponsible for this remarkable diversity of protein expression ingeneral as well as tissue-specific expression of proteins. DNA isinitially transcribed “literally” into pre-messenger RNA (pre-mRNA)comprising introns and exons. The average human protein coding gene is28,000 nucleotides long with 8.8 exons separated by 7.8 introns. Exonsare about 120 nucleotides long while introns are anywhere from100-100,000 nucleotides long. Pre-mRNA is first processed by aspliceosome which recognizes where introns begin and end, removesintrons, and joins exons together to form a mature mRNA that is thentranslated into a protein.

Pre-messenger RNA splicing is an essential process required for theexpression of most genes. Improperly spliced mRNA molecules lead toaltered proteins that cannot function properly, resulting in disease.Alternative splicing errors are known to contribute to cancer and manyneurological diseases, including 13-thalassemia, cystic fibrosis, spinalmuscular atrophy (SMA), growth deficiencies, ataxia, autism, andmuscular dystrophies.

5HT2CR: Prader-Willi syndrome (PWS)

Prader-Willi syndrome (PWS) is a genetic disorder caused by the deletionof paternal copies of several genes on the 15th chromosome located inthe region 15q11-13 leading to deletion of a small nucleolarribonucleoprotein (snoRNA), HBII-52. Deletion of the same region on thematernal chromosome causes Angelman syndrome. The incidence of PWS isabout 1 in 12,000 to 1 in 15,000 live births. Phenotypically,individuals afflicted with PWS typically exhibit significant cognitiveimpairment, hyperphagia often leading to morbid obesity, an array ofcompulsive behaviors, and sleep disorders.

After transcription, nascent or pre-mRNA undergoes a series ofprocessing steps in order to generate a mature mRNA molecule. snoRNAsare non-protein coding RNAs that are 60-300 nucleotides (nt) long andthat function in guiding methylation and pseudouridylation of ribosomalRNA (rRNA), small nuclear RNAs (snRNAs), and transfer RNAs (tRNAs). EachsnoRNA molecule acts as a guide for only one (or two) individualmodifications in a target RNA. In order to carry out the modification,each snoRNA associates with at least four protein molecules in anRNA/protein complex referred to as a small nucleolar ribonucleoprotein(snoRNP). The proteins associated with each RNA depend on the type ofsnoRNA molecule incorporated. The snoRNA molecule contains an antisenseelement (a stretch of 10-20 nucleotides) which are complementary to thesequence surrounding the nucleotide targeted for modification in thepre-RNA molecule. This enables the snoRNP to recognise and bind to thetarget RNA. Once the snoRNP has bound to the target site the associatedproteins are in the correct physical location to catalyse the chemicalmodification of the target base.

The two different types of RNA modification (methylation andpseudouridylation) are directed by two different families of snoRNAs.These families of snoRNAs are referred to as antisense C/D box and H/ACAbox snoRNAs based on the presence of conserved sequence motifs in thesnoRNA. There are exceptions, but as a general rule C/D box membersguide methylation and H/ACA members guide pseudouridylation. HBII-52,also known as SNORD115, belongs to the C/D box class of snoRNAs.

In the human genome, HBII-52 is encoded in a tandemly repeated arraywith another C/D box snoRNA, HBII-85, in the Prader-Willi syndrome (PWS)region of human chromosome 15q11-13. This locus is maternally imprinted,meaning that only the paternal copy of the locus is transcribed.

The snoRNA HBII-52 is exclusively expressed in the brain and is absentin PWS patients. HBII-52 lacks any significant complementarity withribosomal RNAs, but does have an 18 nucleotide region of conservedcomplementarity to exon 5 of serotonin 2C receptor (5-HT2CR) pre-mRNA.snoRNA HBII-52 is an example of an RNA that regulates pre-mRNA splicingby binding to a splice supressor sequence of the 5-HT2CRgene, resultingin enhancement of exon 5b inclusion and the expression of a full-length,functional 5-HT2C receptor.

A recent study showed that these sequences co-varied among species, suchthat differences in nucleotides in one were always matched bycomplementary changes in the other; so that 100% complementarity isalways present (Kishore and Stamm, 2006, Science 311:230-232). Kishoreand Stamm, 2006, Science 311:230-232 also used a minigene construct todemonstrate that interaction of 5-HT2CRand HBII-52 at the consensussequences is critical for appropriate splicing of the 5b exon so that afunctional receptor is generated. When HBII-52 is mutated at sites thatprevent its interaction with 5-HT2C, exon 5a is included and exon 5b isexcluded. The splice variant containing 5a leads to a nonfunctional, outof frame, truncated transcript (Kishore and Stamm, 2006, Science311:230-232).

Dysregulation of serotonergic systems appears to play a role in manycognitive disorders, including depression, autism, and obsessivecompulsive disorder. Although a direct link between dysfunction of5-HT2CRand PWS has yet to be demonstrated, 5-HT2CR knockout mice displayphenotypic characteristics that are remarkably similar to those observedin PWS, including development of hyperphagia-induced obesity. Inpatients with PWS, satiety centers seem to be perturbed, leading toexcessive overeating and obesity. Similarly, in 5-HT2C receptor knockoutmice, obesity develops due to a lack of control of feeding behavior(Nonogaki et al., 1998, Nature Med. 4:1152-1156). 5-HT2CRagonists appearto be effective in inducing satiety (Nilsson, 2006, J. Med. Chem.49:4023-4034). Another notable characteristic of patients with PWS iscompulsive behavior. 5-HT2CR knockout mice also demonstratecompulsive-like behavior (Chou-Green et al., 2003, Physiol. Behav.78:641-649). Interestingly, 5-HT2CR agonists are effective in animalmodels of obsessive-compulsive disorder (OCD); suggesting dysfunction ofthis receptor system could play a role in this disorder (Jenck et al.,1998, Expert. Opin. Invest. Drugs 7:1587-1599; Dunlop et al., 2006, CNSDrug Rev. 12:167-177). The sleep impairment observed in many PWSpatients is also found in the 5-HT2CR knockout mouse (Frank et al.,2002, Neuropsychopharmacology 27:869-873). These mice also exhibitedreduced hippocampal-dependent learning and deficits in hippocampalsynaptic plasticity that appears to be critical in learning and memory(Tecott et al., 1998, Proc. Natl. Acad. Sci. 95:15026-15031). Thus5-HT2C receptor knockouts may replicate some of the cognitive deficitsfound in PWS. 5-HT2CR knockout mice therefore share many, but not all(e.g., failure to thrive, which may be mediated by HBII-85 (Ding et al.,2005, Mamm. Genome 16:424-431)), critical phenotypes with PWS patients.

AMPA Receptor: Excitotoxicity, Seizure, and Amyotrophic LateralSclerosis (ALS)

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (alsoknown as AMPA receptor, AMPAR, or quisqualate receptor) is anon-NMDA-type ionotropic transmembrane receptor for glutamate in thecentral nervous system (CNS). Postsynaptic ion channels activated byglutamate include NMDA (N-methyl-D-aspartic acid)-type glutamatechannels, which are highly Ca²⁺ permeable, and AMPA-type glutamatechannels, which mediate the majority of rapid excitatoryneurotransmission. AMPA channels are homo- or hetero-oligomericassemblies composed of various combinations of four possible subunits,GluR1, GluR2, GluR3 and GluR4. The Ca²⁺ conductance of AMPA receptorsdiffers markedly according to whether the GluR2 subunit is present ornot and whether it has undergone post-transcriptional RNA editing at theQ/R site. AMPA receptors that contain at least one Q/R edited GluR2subunit are Ca²⁺ impermeable. These properties of GluR2 are generated byRNA editing at the Q/R site in the putative second transmembrane domain(M2), during which a glutamine (Q) codon is replaced by an arginine (R)codon (Seeburg et al., 2001, Brain Res. 907:233-243). It is thought thatarginine in the pore of the channel impedes Ca²⁺ permeation. Analyses ofadult rat, mouse, and human brains have demonstrated that almost allGluR2 mRNA in neurons is edited. In contrast, the Q/R site of GluR1,GluR3 and GluR4 subunits are always unedited, and glutamine remains atthis crucial position. Therefore, AMPA receptors lacking a Q/R editedGluR2 subunit or lacking GluR2 altogether are highly Ca²⁺ permeable(Kawahara and Kwak, 2005, ALS Other Motor Neuron Disord 6:131-144;Seeburg et al., 2001, Brain Res. 907:233-243).

Alternative splicing of the GluRs plays a critical role in AMPA receptorphysiology, influencing sensitivity to glutamate, kinetics of channeldesensitization, and intracellular trafficking. Two specificalternatively spliced variants of all GluRs called “flip” and “flop” arenormally expressed in the CNS. These consist of 115 base pair exons thatconstitute the flip/flop cassette (Sommer et al., 1990, Science249:1580-1585) and encode part of the extracellular segment thatprecedes the fourth transmembrane domain. This domain appears tomodulate receptor desensitization and channel conductance (Mosbacher etal., 1994, Science 266:1059-1062). Generally, the AMPA “flip” variantsare resistant to desensitization, whereas the “flop” variants arereadily desensitized, although the kinetic difference depends on thesubunit and, for heteromeric channels, on subunit compositions(Grosskreutz et al., 2003, Eur. J. Neurosci. 17:1173-1178; Koike et al.,2000, J. Neurosci. 25:199-207; Mosbacher et al., 1994; Sommer et al.,1990, Science 249:1580-1585). The extracellular flip/flop region mayalso interact with ER luminal proteins to regulate trafficking of AMPAreceptors, with flip isoforms inserted into the cell membrane and flopisoforms trapped internally (Coleman et al., 2006, J. Neurosci.26:11220-11229), although this has yet to be confirmed in neurons.Together these data show that when flip/flop ratio of GluR1, GluR3 andGluR4 is elevated, neurons are more excitable and show greater Ca²⁺conductance.

In motor neurons (MNs) it has been consistently demonstrated that AMPAreceptor desensitization significantly impacts the shape of theglutamatergic synaptic response, as well as robustly regulating networkactivity (Ballerini et al., 1995, Eur. J. Neurosci. 7:1229-1234; Funk etal., 1995, J. Neurosci. 15:4046-4056). In addition, studies in severaldifferent brain regions have found that AMPA receptor desensitizationhas potent effects on baseline evoked and spontaneous synaptic events(Akopian and Walsh, 2007, J. Physiol. 580:225-240; Atassi andGlavinovic, 1999, Pflugers Arch. 437:471-478; Xia et al., 2005, J.Pharmacol. Exp. Ther. 313:277-285), although this is controversial,especially in hippocampus (Arai and Lynch, 1998, Brain Res. 799:230-234;Hjelmstad et al., 1999, J. Neurophysiol. 81:3096-3099). Further, AMPAreceptor desensitization has been shown to be critical in shaping thesynaptic response under conditions of higher frequency activity bystrongly regulating synaptic integration (Arai and Lynch, 1998, BrainRes. 799:235-234; Chen et al., 2002, Neuron 33:779-788). Prolonging AMPAchannel desensitization can also generate excessive networksynchronization, leading to paroxysmal bursting that may interfere withnormal network function (Funk et al., 1995, J. Neurosci. 15:4046-4056;Pelletier and Hablitz, 1994, J. J. Neurophysiol. 72:1032-1036; Qiu etal., 2008, J. Neurosci. 28:3567-3576). Thus, it is not surprising thatreducing AMPA receptor desensitization profoundly increasesexcitotoxicity induced by glutamate and AMPA.

In spinal MNs, as well as in hippocampus and cerebellar granule cells,treatment with AMPA alone does not induce neurotoxicity. However, AMPAcombined with cyclothiazide, which greatly reduces AMPA receptordesensitization, leads to neuronal cell death (Carriedo et al., 2000, J.Neurosci. 20:240-250; May and Robison, 1993, J. Neuroschem.60:1171-1174; Puia et al., 2000, Prog. Neuropsychopharm. Biol.Psychiatr. 24:1007-1015). AMPA-mediated neurotoxicity is also amplifiedby cyclothiazide in cerebellar purkinje cells (Brorson et al., 1995, J.Neurosci. 15:4515-4524) and cortical neurons (Jensen et al., 1998,Neurochem. Int. 32:505-513). Further, in HEK293 cells, AMPA inducesexcitotoxicity when flip but not flop GluR isoforms are expressed(fizuka et al., 2000, Eur. J. Neurosci. 12:3900-3908). AMPA receptordesensitization can also protect against NMDA receptor mediatedexcitotoxicity (Jensen et al., 1998, Neurochem. Int. 32:505-513;Zorumski et al., 1990, Neuron 5:61-66). Finally, decreases in AMPAreceptor desensitization have been proposed to play a role inexcitotoxicity after traumatic brain injury (Goforth et al., 1999, J.Neurosci. 19:7367-7374). Thus, AMPA receptor desensitization plays acritical role in normal neuronal function and excitotoxicity.

Emerging evidence supports the idea that Ca²⁺-permeable AMPA channels,which are highly expressed on MNs, are key contributors to injury of MNsin amyotrophic lateral sclerosis (ALS) (Corona et al., 2007, ExpertOpin. Ther. Targets 11:1415-1428; Van Den et al., 2006, Biochem.Biophys. Acta. 1762:1068-1082). Compared to most cell types, MNs haverelatively poor capacity to buffer Ca²⁺, due to reduced levels of Ca²⁺binding proteins including calbindin and parvalbumin (Alexianu et al.,1994, Ann. Neurol. 36:846-858; Ince et al., 1993, Neuropathol. Appl.Neurobiol. 19:291-299; Palecek et al., 1999, J. Physiol. 520 pt 2:485-502). It appears that spinal MNs of ALS mice have even fewer ofthese Ca²⁺-binding proteins (Siklos et al., 1998, J. Neuropathol. Exp.Neurol. 57:571-587). Amplifying that point, recent studies have shownthat G93A ALS mice interbred with mice overexpressing parvalbumin showeda delayed onset of motor disease (Beers et al., 2001, J. Neurochem.79:499-509). According to a speculative model of glutamate-mediatedexcitotoxicity involving AMPA channels in ALS, Ca²⁺ influx throughCa²⁺-permeable AMPA channels is not adequately buffered in MNs and endsup accumulating in mitochondria. High Ca²⁺ is toxic to mitochondria,causing generation of apoptotic mediators such as ROS and cytochrome c,as well as opening of a permeability transition pore through whichapoptotic mediators are released. It is thought that these mitochondrialfactors are released from MNs and exert deleterious effects on glutamatetransporters on adjacent astrocytes. Astrocytic glutamate transportersare responsible for taking up synaptic glutamate, and when they arecompromised, glutamate accumulates in the synaptic region. The glutamatetransporter with the most functional significance in this context isEAAT2/GLT-1, as it is widely expressed in astrocytes throughout the CNSand as it has the highest affinity for glutamate. In over −65% of ALScases, and in ALS mice, EAAT2 activity in the cortex and spinal cord iscompromised (Van Den et al., 2006, Biochem. Biophys. Acta.1762:1068-1082). Thus, in this model, increased glutamate then furtherstimulates more Ca²⁺ influx though AMPA channels causing a feed-forwardcycle that ultimately leads to too much Ca²⁺ in MNs. This sets intomotion a cascade that leads by unknown mechanisms to MN cell death.

There is also evidence that decreased desensitization of AMPA channels,due to increased flip/flop expression ratio, may exacerbate glutamateexcitotoxicity in ALS. In spinal MNs of ALS subjects, the level of theAMPA receptor flip variants was found to be significantly elevatedrelative to that of the flop isoforms (Tomiyama et al., 2002, Synapse45:245-249). Although this work from a highly published neuroanatomygroup is the only study thus far to examine flip/flop isoforms in spinalcord of ALS patients, the findings were quite compelling. They observeda 41-66% decrease in the flop isoforms of GluR1-3 only in the ventralhorn (layer IX), where MN soma are localized. Further, they providedevidence that their transcript labeling was restricted to MN soma.Unfortunately, flip/flop protein levels were not examined, sincespecific antibodies for flip and flop isoforms of GluRs do not exist. Aremarkably similar change in AMPA receptor flip/flop ratios wasindependently observed in MNs from G93A SOD 1 ALS mice (Spalloni et al.,2004, Neurobiol. Dis. 15:340-350). This study showed increased flipisoforms, especially GluR3 and GluR4, and thus dramatic increases inflip to flop ratios. Interestingly, these changes were specific to miceoverexpressing mutant SOD1 but were not found in mice overexpressingnormal human SOD1. Further, electrophysiological studies demonstratedreduced desensitization of AMPA currents in MNs of G93A transgenicscompared to control and SOD1 transgenics, as well as robust increases inblockade of desensitization by cyclothiazide. Both of these propertiesare characteristic of increased flip isoforms (Partin et al., 1994, Mol.Pharm. 46:129-138; Sommer et al., 1990, Science 249:1580-1585). Inaddition, spontaneous glutamatergic synaptic events are prolonged due toincreased decay times in MNs of G93A ALS mice compared to control andSOD1 transgenics, also consistent with an increase in flip isoforms(Pieri et al., 2003, Neurosci. 122:47-58). Together, these studiesindicate that aberrant flip-flop ratios are present in MNs of ALSindividuals, and that these changes are replicated in a mouse model ofthe disease. These data strongly implicate a contribution of aberrantflip-flop levels of AMPA channels to MN excitotoxicity in ALS.Specifically, MNs with high levels of Ca²⁺-permable AMPA receptors(Kawahara et al., 2004, Nature 427:801), and especially membrane boundnon-desensitizable flip isoforms, permit enhanced postsynaptic Ca²⁺influx in response to a given glutamate load (FIG. 2).

Increases in the flip to flop ratio in adult hippocampus have also beenreported after seizures. This recapitulation of the immature phenotypeafter seizures is seen for many other neurotransmitter related proteins(Brooks-Kayal et al., 1998). In rat hippocampus, the flip variant ofboth GluR1 and GluR2 is increased after seizures induced by tetanustoxin (Rosa et al., 1999, Epilepsy Res. 36:243-251) and kindling(Kamphuis et al., 1992, Neurosci. Lett. 148:51-54; Kamphuis et al.,1994, nature 448:39-43). In hippocampal tissue from humans withepilepsy, increases in flip-flop ratios have also been reported. TheGluR1 flip variant is increased in hippocampal astrocytes, as assessedboth functionally with electrophysiology and at the transcript levelwith single-cell real time PCR (Seifert et al., 2004, J. Neurosci.24:1996-2003). In hippocampal neurons, expression of the GluR1 flipvariant is increased in CA1 after seizures (Eastwood et al., 1994,Neuroreport 5:1325-1328; de Lanerolle et al., 1998, Eur. J. Neurosci.10:1687-1703). While the flop variant is found in CA3 and dentate innon-epileptic hippocampus (Eastwood et al., 1994, Neuroreport5:1325-1328), in tissue from patients with TLE the flop variant of GluR1is found only in the dentate (de Lanerolle et al., 1998, Eur. J.Neurosci. 10:1687-1703). Thus flop appears to be downregulated in CA3 inepileptic hippocampus. The increase in flip to flop ratios in epileptichippocampus would increase synaptic gain and could contribute topost-seizure hyperexcitability.

Aph1B: Alzheimer's Disease

Alzheimer's Disease (AD) is a common neurodegenerative disorder andresults in a severe decline in cognition, and ultimately dementia,especially in the aged population. Progression of the disease is linkedto the characteristic deposition of β-amyloid and tau neurofibrillarytangles (NTs).

Compelling evidence shows that amyloid-beta peptide (Aβ) contributes tothe etiology of AD. Aβ is a 38-43 amino acid peptide that is produced inneurons by the sequential proteolytic cleavage of APP by β-secretase andγ-secretase, the latter step yielding isoforms Aβ40 and Aβ42. Aβ42appears to be the most highly amyloidogenic isoform. In humans,γ-secretase complexes are heterogeneous, comprised of two presenilingenes (PS1 and PS2), along with Aph1A (long or short isoforms) and Aph1B(Shirotani et al., 2004, J. Biol. Chem. 279:41340-41345).

Gamma-secretase is a tri-partite protein complex composed of presenilin,nicastrin, and ApH1. ApH1 is composed of both Aph1A and Aph1B.Transgenic elimination of Aph1B blocked the processing of amyloidprecursor protein (APP) to A-beta, but did not effect the processing ofother non-amyloidal substrates (Serneels et al., 2009, Science324:639-642).

A common understanding about AD is that APP processing sequentially byBACE then gamma-secretase, results in the production of Abeta42 amongother isoforms. The Abeta42 isoform, which is the direct product ofgamma-secretase cleavage is thought to be especially harmful, first as asoluble factor that impairs cognition and later in the production ofamyloid plaques that may further enhance disease progression. Therefore,an intense search for compounds that reduce the activity ofgamma-secretase is underway. Unfortunately, in addition to activelycleaving APP, gamma-secretase also cleaves a number of other importantnon-amyloidal substrates, such as Notch. Thus, there is an urgent needfor improved compounds that significantly reduce gamma-secretaseproduction of Aβ-42 in the brain, without affecting its cleavage ofother non-APP substrates.

O-GlcNAcase (OGA): Alzheimer's Disease

Levels of N-acetyl-D-glucosamine (O-GlcNAc) modification of proteins areknown to be reduced throughout the brains of Alzheimer's Disease (AD)patients due to low glucose availability, and this global alteration isthought to be pathological in AD progression (Fischer, 2008, NatureChem. Biol. 4:448-449). Dynamic cycling of O-GlcNAc is regulated byaddition through N-acetyl-D-glucosamine polypeptidyltransferase (OGT)and removal by O-GlcNAcase (OGA). Removal of O-GlcNAc from proteins byOGA may be involved in controlling multiple cellular pathways. OGA hasbeen shown to mediate transcriptional activation both by directlymodifying the transcriptome and by preventing the recycling oftranscription factors in the nucleus (Bowe et al., 2006, Mol. Cell.Biol. 26:8539-8550). Additionally, OGA has been implicated in chromatinremodeling and transcriptional repression via interactions withOGT/histone deacetylase (HDAC) complexes and or C-terminal histoneacetyltransferase (HAT) activity (Lazarus et al., 2009, Int. J. Biochem.Cell Biol. 41:2134-2146; Whisenhunt et al., 2006, Glycobiol.16:551-563). There is also evidence that phosphorylation andO-GlcNAcylation exist in dynamic equilibrium. Serine/threonine residuesthat otherwise may be phosphorylated by serine/threonine kinases can beinstead O-GlcNAc modified, as is the case with tau (Yuzwa et al., 2008,Nat. Chem. Biol. 4:483-490). Further evidence indicates thatO-GlcNAcylation of tau can cause trafficking and retention of tau in thenucleus (Guinez et al., 2005, Int. J. Biochem. Cell Biol. 37:765-774)Importantly to AD pathology, low levels of O-GlcNAc on tau may allow fortau hyperphosphorylation, which leads to neurofibrillary tangle (NT)formation. Thus alteration of brain glycosylation will have effects onmultiple pathways.

HER3: Cancer

About 25% of breast cancers involve overexpression of the HER2, withhighly aggressive metastasis, and poor clinical prognosis. Herceptinshows some success against HER2 overexpressing breast cancer cells(HOBCsa), and tyrosine kinase inhibitors (TKIs) have shown promise inearly clinical trials. However, HOBCs show remarkable acquiredresistance to current drugs. Recent studies have shown HER3 isoverexpressed in HOBCs and exerts a critical role in tumorogenesis,metastasis, and acquisition of resistance to TKIs (Baselga, J. & Swain,S. M. (2009) Novel anticancer targets: revisiting ERBB2 and discoveringERBB3. Nat Rev Cancer 9:463-475). For EGFRs, dimerization andtransactivation by tyrosine kinase is essential for signaling activity.Although HER3 lacks intrinsic tyrosine kinase activity, the most potentEGFR activated dimers are heterodimers between HER2 and HER3, leading topotent HER3-mediated TKI resistance via activation of the PI3K-Aktpathway (Baselga et al., 2009, Nat Rev Cancer 9:463-475). Since the lossof HER3 function ameliorates the transforming capabilities of HER2,there is a pressing need for new drugs against HER3 for treating breastcancer.

Forkhead Box Protein M1 (FOXM1): Anti-Tumor

Forkhead box protein M1 (FOXM1) is a protein that is encoded by the FOX1gene and is a member of the FOX family of transcription factors. FOXM1is known to play a key role in cell cycle progression. There are threeFOXM1 isoforms, A, B and C. Isoform FOXM1A has been shown to be a genetranscriptional repressor whereas the remaining isoforms (B and C) areboth transcriptional activators. Hence, it is not surprising that FOXM1Band C isoforms have been found to be upregulated in human cancers(Wiestra et al., 2007, Biol. Chem. 388 (12): 1257-74.

The exact mechanism of FOXM1 in cancer formation remains unknown. It isthought that upregulation of FOXM1 promotes oncogenesis through abnormalimpact on its multiple roles in cell cycle and chromosomal/genomicmaintenance. FOXM1 overexpression is involved in early events ofcarcinogenesis

FOXM1 gene is now known as a human proto-oncogene. Abnormal FOXM1upregulation was subsequently found in the majority of solid humancancers including liver (The et al., 2002, Cancer Res. 62: 4773-80)breast (Wonsey et al., 2005, Cancer Res. 65 (12): 5181-9), lung (Kim etal., 2006, Cancer Res. 66 (4): 2153-61), prostate (Kalin et al., 2006,Cancer Res. 66 (3): 1712-20; cervix of uterus (Chan et al., 2008, J.Pathol. 215 (3): 245-52), colon (Douard et al., 2006, Surgery 139 (5):665-70), pancreas (Wang et al., 2007, Cancer Res. 67 (17): 8293-300),and brain (Liu et al., 2006, Cancer Res. 66 (7): 3593-602).

Cyclophilin D: ALS, Hepatitis B Viral Infection, and Liver Cancer

Cyclophilin D (CypD) is a protein located in the matrix of themitochondria, and is one of the components of the mitochondrialpermeability transition pore (MPTP). Under conditions of oxidativestress, the MPTP becomes extremely permeable to the influx of calciumions, therein causing mitochondrial swelling eventually leading to cellapoptosis. Targeting the MPTP/CypD complex in hepatitis B virus (HBV)infected hepatocytes using the non-specific CypD inhibitor, CyclosporinA, inhibits HBV replication (Waldemeier et al., 2003, Current MedicinalChemistry 10:1485-1506). In addition, when used in patients withneurodegerative diseases, Cyclosporin A exhibits cytoprotective effectsby way of blocking the opening of the MPTP. Although shown to beefficacious, Cyclosporin A is an immunosuppressive drug, and can alsobind non-specifically to other cyclophilins, therefore causingoff-target effects. Inhibition of CypD expression using siRNA has beenexamined as a potential cardioprotective therapy (Kato et al., 2009,Cardiovasc. Res. 83:335-344). However SMOs have a therapeutic advantageover siRNA in that unlike siRNA, SMOs do not affect transcriptdegradation through recruitment of RNAase H which can cause immunereactions and other off target effects.

There is presently no known cure for PWS, ALS, AD or any of a number ofother diseases that result from aberrant pre-mRNA splicing. There is aneed in the art for the development of more selective and efficacioustherapeutic agents for the treatments of various diseases and conditionsaffected or mediated by 5HT2CR, GluRs, OGA, Aph1B, FOXM1, ERBB3, andCypD. In addition, there are a number of diseases where alteringpre-mRNA splicing may have a positive therapeutic effect even when thatgene is not directly affected by the pathogenesis of the disease.Accordingly, there is an urgent need in the art for compositions andmethods related to pre-mRNA splicing as it affects various diseases anddisorders. The present invention fills this need.

SUMMARY OF THE INVENTION

In one embodiment, the present invention comprises a method ofmodulating splicing of a pre-mRNA, the method comprising contacting acell with an effective amount of a splice modulating oligonucleotide(SMO), where the SMO specifically binds to a complementary sequence on apre-mRNA in at least one of the group consisting of an intron-exonsplice site, an exonic splice enhancer (ESE) site, and an intronicsplice enhancer (ISE) site, where when the SMO specifically binds to thecomplementary sequence, the exon adjacent to the intron-exon boundary isexcluded from the resulting mRNA. In one aspect, the resulting mRNAencodes a protein selected from the group consisting of a glutamateactivated AMPA receptor subunit (GluR), OGA, Aph1B, FOXM1, HER3, andCypD. In another aspect, the GluR is selected from the group consistingof GluR1, GluR2, GluR3, GluR4 and any combination thereof.

In another embodiment, the present invention comprises a method ofmodulating splicing of a pre-mRNA, the method comprising contacting acell with an effective amount of a splice modulating oligonucleotide(SMO), where the SMO specifically binds to a complementary sequence on apre-mRNA in at least one of the group consisting of an intron-exonsplice site, an exonic splice suppressor (ESS) site, and an intronicsplice suppressor (ISS) site, where when the SMO specifically binds tothe complementary sequence, the exon adjacent to the intron-exonboundary is included in the resulting mRNA. In one aspect, the resultingmRNA encodes a 5-HT2C receptor.

In still another embodiment, the present invention comprises a method oftreating a subject afflicted with a 5-HT2CR splicing defect, the methodcomprising administering a splice modulating oligonucleotide (SMO) tothe subject, where an effective amount of the SMO contacts a cell sothat the SMO specifically binds to a complementary sequence on apre-mRNA, where when the SMO specifically binds to the complementarysequence, exon 5b is included in the resulting mRNA encoding afull-length, functional 5-HT2C receptor, and where the SMO increasesexpression of a full-length, functional 5-HT2C receptor in the subjectand treats the subject afflicted with a 5-HT2CR splicing defect. In oneaspect, the SMO is an isolated nucleic acid sequence selected from thegroup consisting of SEQ ID NOs. 1-56.

In another embodiment, the present invention comprises a method oftreating a subject afflicted with Prader-Willi Syndrome (PWS), themethod comprising administering a splice modulating oligonucleotide(SMO) to the subject, where an effective amount of the SMO contacts acell so that the SMO specifically binds to a complementary sequence on apre-mRNA, where when the SMO specifically binds to the complementarysequence, exon 5b is included in the resulting mRNA encoding afull-length, functional 5-HT2C receptor, and where the SMO increasesexpression of the full-length, functional 5-HT2C receptor in the subjectand treats the subject afflicted with PWS. In one aspect, the SMO is anisolated nucleic acid sequence selected from the group consisting of SEQID NOs. 1-56.

In yet another embodiment, the present invention comprises, a method oftreating a subject afflicted with hyperphagia, the method comprisingadministering a splice modulating oligonucleotide (SMO) to the subject,where an effective amount of the SMO contacts a cell so that the SMOspecifically binds to a complementary sequence on a pre-mRNA, where whenthe SMO specifically binds to the complementary sequence, exon 5b isincluded in the resulting mRNA encoding a full-length, functional 5-HT2Creceptor, and where the SMO increases expression of the full-length,functional 5-HT2C receptor in the subject and treats the subjectafflicted with hyperphagia. In one aspect, the SMO is an isolatednucleic acid sequence selected from the group consisting of SEQ ID NOs.1-56.

In still another embodiment, the present invention comprises a method oftreating a subject afflicted with symptoms of obsessive-compulsivedisorder, the method comprising administering a splice modulatingoligonucleotide (SMO) to the subject, where an effective amount of theSMO contacts a cell so that the SMO specifically binds to acomplementary sequence on a pre-mRNA, where when the SMO specificallybinds to the complementary sequence, exon 5b is included in theresulting mRNA encoding a full-length, functional 5-HT2C receptor, andwhere the SMO increases expression of the full-length, functional 5-HT2Creceptor in the subject and treats the subject afflicted with symptomsof obsessive-compulsive disorder. In one aspect, the SMO is an isolatednucleic acid sequence selected from the group consisting of SEQ ID NOs.1-56.

In another embodiment, the present invention comprises a method ofincreasing expression of a transmembrane neuronal receptor in a subject,the method comprising contacting a cell with an effective amount of asplice modulating oligonucleotide (SMO), where the SMO specificallybinds to a complementary sequence on a pre-mRNA in at least one of anintron-exon splice site, an exonic splice suppressor (ESS) site, and anintronic splice suppressor (ISS) site, where when the SMO specificallybinds to the complementary sequence, the exon adjacent to theintron-exon boundary is included in the resulting mRNA encoding afull-length, functional transmembrane neuronal receptor, and where theSMO increases expression of the full-length, functional transmembraneneuronal receptor. In one aspect, the transmembrane neuronal receptor isa 5-HT2C receptor. In another aspect, the subject is afflicted with adisease or disorder selected from the group consisting of PWS, AngelmanSyndrome, hyperphagia induced obesity, obsessive/compulsive disorder,depression, psychotic depression, major depressive disorder, bipolardisorder, sleep impairment, autism, schizophrenia, Parkinson's disease,drug addiction, spinal cord injury, traumatic brain injury, neuropathicpain, diabetes, and Alzheimer's disease.

In still another embodiment, the present invention comprises a method ofincreasing expression of a transmembrane neuronal receptor in a subject,the method comprising contacting a cell with an effective amount of asplice modulating oligonucleotide (SMO), where the SMO specificallybinds to a complementary sequence on a pre-mRNA in at least one of thegroup consisting of an intron-exon splice site, an exonic spliceenhancer (ESE) site, and an intronic splice enhancer (ISE) site, wherewhen the SMO specifically binds to the complementary sequence, the exonadjacent to the intron-exon boundary is excluded from the resulting mRNAencoding the transmembrane neuronal receptor, and where the SMOincreases expression of the transmembrane neuronal receptor. In oneaspect, the transmembrane neuron receptor is a glutamate activated AMPAreceptor subunit (GluR) selected from the group consisting of GluR1,GluR2, GluR3, GluR4, and any combination thereof.

In another embodiment, the present invention comprises a method oftreating a subject afflicted with amyotrophic lateral sclerosis (ALS),the method comprising administering a splice modulating oligonucleotide(SMO) to the subject, where an effective amount of the SMO contacts acell so that the SMO specifically binds to a complementary sequence on apre-mRNA in at least one of an intron-exon splice site, an exonic spliceenhancer (ESE) site, and an intronic splice enhancer (ISE) site, wherewhen the SMO specifically binds to the complementary sequence, the exonadjacent to the intron-exon boundary is excluded from the resulting mRNAencoding a GluR, where the GluR is selected from the list consisting ofGluR1, GluR2, GluR3, GluR4, and any combination thereof, where the SMOdecreases expression of the flip isoform of the GluR in the subject andtreats the subject afflicted with ALS. In one aspect, the SMO is anisolated nucleic acid sequence selected from the group consisting of SEQID NOs. 57-526.

In yet another embodiment, the present invention comprises a method oftreating a subject afflicted with epilepsy the method comprisingadministering a splice modulating oligonucleotide (SMO) to the subject,where an effective amount of the SMO contacts a cell so that the SMOspecifically binds to a complementary sequence on a pre-mRNA in at leastone of an intron-exon splice site, an exonic splice enhancer (ESE) site,and an intronic splice enhancer (ISE) site, where when the SMOspecifically binds to the complementary sequence, the exon adjacent tothe intron-exon boundary is excluded from the resulting mRNA encoding aGluR, where the GluR is selected from the list consisting of GluR1,GluR2, GluR3, GluR4, and any combination thereof, where the SMOdecreases expression of the flip isoform of the GluR in the subject andtreats the subject afflicted with epilepsy. In one aspect, the SMO is anisolated nucleic acid sequence selected from the group consisting of SEQID NOs. 57-526.

In another embodiment, the present invention comprises a method oftreating a subject afflicted with Alzheimer's Disease (AD), the methodcomprising administering a splice modulating oligonucleotide (SMO) tothe subject, where an effective amount of the SMO contacts a cell sothat the SMO specifically binds to a complementary sequence on apre-mRNA in at least one of an intron-exon splice site, an exonic spliceenhancer (ESE) site, and an intronic splice enhancer (ISE) site, wherewhen the SMO specifically binds to the complementary sequence, the exonadjacent to the intron-exon boundary is excluded from the resultingmRNA, where the exon is exon 8 of the mRNA encoding O-GlcNAcase (OGA),where the SMO increases expression of OGAΔ8 in the subject and treatsthe subject afflicted with AD. In one aspect the SMO is an isolatednucleic acid sequence selected from the group consisting of SEQ ID NOs.527-611.

In still another embodiment, the present invention comprises a method oftreating a subject afflicted with Alzheimer's Disease (AD), the methodcomprising administering a splice modulating oligonucleotide (SMO) tothe subject, where an effective amount of the SMO contacts a cell sothat the SMO specifically binds to a complementary sequence on apre-mRNA in at least one of an intron-exon splice site, an exonic splicesuppressor (ESS) site, and an intronic splice suppressor (ISS) site,where when the SMO specifically binds to the complementary sequence, theintron adjacent to the intron-exon boundary is included in the resultingmRNA, where the intron is intron 10 of the mRNA encoding O-GlcNAcase(OGA), and where the SMO increases expression of a truncated OGA protein(OGA10t) in the subject and treats the subject afflicted with AD. In oneaspect, the SMO is an isolated nucleic acid sequence selected from thegroup consisting of SEQ ID NOs. 612-661.

In another embodiment, the present invention comprises a method oftreating a subject afflicted with Alzheimer's Disease (AD), the methodcomprising administering a splice modulating oligonucleotide (SMO) tothe subject, where an effective amount of the SMO contacts a cell sothat the SMO specifically binds to a complementary sequence on apre-mRNA in at least one of the group consisting of an intron-exonsplice site, an exonic splice enhancer (ESE) site, and an intronicsplice enhancer (ISE) site, where when the SMO specifically binds to thecomplementary sequence, the exon adjacent to the intron-exon boundary isexcluded from the resulting mRNA, where the exon is exon 4 of the mRNAencoding Aph1B, where the SMO increases expression of Aph1BΔ4 in thesubject and treats the subject afflicted with AD. In one aspect, the SMOis an isolated nucleic acid sequence selected from the group consistingof SEQ ID NOs. 662-728.

In yet another embodiment, the preset invention comprises a method oftreating a subject afflicted with a cancer, the method comprisingadministering a splice modulating oligonucleotide (SMO) to the subject,where an effective amount of the SMO contacts a cell so that the SMOspecifically binds to a complementary sequence on a pre-mRNA in at leastone of the group consisting of an intron-exon splice site, an exonicsplice enhancer (ESE) site, and an intronic splice enhancer (ISE) site,where when the SMO specifically binds to the complementary sequence, theexon adjacent to the intron-exon boundary is excluded from the resultingmRNA, where the exon is exon 3 of the mRNA encoding HER3, and where theSMO increases expression of a HER3Δ3 in the subject and treats thesubject afflicted with cancer. In one aspect, the SMO is an isolatednucleic acid sequence selected from the group consisting of SEQ ID NOs.729-802. In another aspect, the cancer is selected from the groupconsisting of breast cancer, liver cancer, lung cancer, prostate cancer,cervical cancer, colon cancer, pancreatic cancer, and brain cancer.

In still another embodiment, the present invention comprises a method oftreating a subject afflicted with a cancer, the method comprisingadministering a splice modulating oligonucleotide (SMO) to the subject,where an effective amount of the SMO contacts a cell so that the SMOspecifically binds to a complementary sequence on a pre-mRNA in at leastone of the group consisting of an intron-exon splice site, an exonicsplice suppressor (ESS) site, and an intronic splice suppressor (ISS)site, where when the SMO specifically binds to the complementarysequence, the intron adjacent to the intron-exon boundary is included inthe resulting mRNA, where the intron is intron 3 of the mRNA encodingHER3, and where the SMO increases expression of a truncated HER3 proteinin the subject and treats the subject afflicted with cancer. In oneaspect, the method of claim 32, where the SMO is an isolated nucleicacid sequence selected from the group consisting of SEQ ID NOs. 729-802.In another aspect, the cancer is selected from the group consisting ofbreast cancer, liver cancer, lung cancer, prostate cancer, cervicalcancer, colon cancer, pancreatic cancer, and brain cancer.

In another embodiment, the present invention comprises a method oftreating a subject afflicted with a cancer, the method comprisingadministering a splice modulating oligonucleotide (SMO) to the subject,where an effective amount of the SMO contacts a cell so that the SMOspecifically binds to a complementary sequence on a pre-mRNA in at leastone of the group consisting of an intron-exon splice site, an exonicsplice enhancer (ESE) site, and an intronic splice enhancer (ISE) site,where when the SMO specifically binds to the complementary sequence, theexon adjacent to the intron-exon boundary is excluded from the resultingmRNA, where the exon is exon 11 of the mRNA encoding HER3, and where theSMO increases expression of a HER3Δ11 in the subject and treats thesubject afflicted with cancer. In one aspect, the SMO is an isolatednucleic acid sequence selected from the group consisting of SEQ ID NOs.803-813. In another aspect, the cancer is selected from the groupconsisting of a breast cancer, liver cancer, lung cancer, prostatecancer, cervical cancer, colon cancer, pancreatic cancer, and braincancer.

In yet another embodiment, the present invention comprises a method oftreating a subject afflicted with a cancer, the method comprisingadministering a splice modulating oligonucleotide (SMO) to the subject,where an effective amount of the SMO contacts a cell so that the SMOspecifically binds to a complementary sequence on a pre-mRNA in at leastone of the group consisting of an intron-exon splice site, an exonicsplice enhancer (ESE) site, and an intronic splice enhancer (ISE) site,where when the SMO specifically binds to the complementary sequence, theexon adjacent to the intron-exon boundary is excluded from the resultingmRNA encoding FOXM1, and where the SMO increases expression of a FOXM1Δ3or FOXM1Δ6 in the subject and treats the subject afflicted with cancer.In one aspect, the SMO is an isolated nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs. 919-1090.

In still another embodiment, the present invention comprises a method oftreating a subject afflicted with a hepatitis B virus (HBV) infection,the method comprising administering a splice modulating oligonucleotide(SMO) to the subject, where an effective amount of the SMO contacts acell so that the SMO specifically binds to a complementary sequence on apre-mRNA in at least one of the group consisting of an intron-exonsplice site, an exonic splice enhancer (ESE) site, and an intronicsplice enhancer (ISE) site, where when the SMO specifically binds to thecomplementary sequence, the exon adjacent to the intron-exon boundary isexcluded from the resulting mRNA, where the exon is exon 1 of the mRNAencoding CypD, and where the SMO increases expression of a CypDΔ1 in thesubject and treats the subject afflicted with an HBV infection. In oneaspect, the SMO is an isolated nucleic acid sequence selected from thegroup consisting of SEQ ID NOs. 814-857.

In yet another embodiment, the present invention comprises a method oftreating a subject afflicted with a hepatitis B virus (HBV) infection,the method comprising administering a splice modulating oligonucleotide(SMO) to the subject, where an effective amount of the SMO contacts acell so that the SMO specifically binds to a complementary sequence on apre-mRNA in at least one of the group consisting of an intron-exonsplice site, an exonic splice enhancer (ESE) site, and an intronicsplice enhancer (ISE) site, where when the SMO specifically binds to thecomplementary sequence, the exon adjacent to the intron-exon boundary isexcluded from the resulting mRNA, where the exon is exon 3 of the mRNAencoding CypD, and where the SMO increases expression of a CypDΔ3 insubject and treats the subject afflicted with an HBV infection. In oneaspect, the SMO is an isolated nucleic acid sequence selected from thegroup consisting of SEQ ID NOs. 858-918.

In another embodiment, the present invention comprises a method oftreating a subject afflicted with a cancer, the method comprisingadministering a splice modulating oligonucleotide (SMO) to the subject,where an effective amount of the SMO contacts a cell so that the SMOspecifically binds to a complementary sequence on a pre-mRNA in at leastone of the group consisting of an intron-exon splice site, an exonicsplice enhancer (ESE) site, and an intronic splice enhancer (ISE) site,where when the SMO specifically binds to the complementary sequence, theexon adjacent to the intron-exon boundary is excluded from the resultingmRNA, where the exon is exon 1 of the mRNA encoding CypD, and where theSMO increases expression of a CypDΔ1 in the subject and treats thesubject afflicted with cancer. In one aspect, the SMO is an isolatednucleic acid sequence selected from the group consisting of SEQ ID NOs.814-857. In another aspect, the cancer is a liver cancer.

In still another embodiment, the present invention comprises a method oftreating a subject afflicted with a cancer, the method comprisingadministering a splice modulating oligonucleotide (SMO) to the subject,where an effective amount of the SMO contacts a cell so that the SMOspecifically binds to a complementary sequence on a pre-mRNA in at leastone of the group consisting of an intron-exon splice site, an exonicsplice enhancer (ESE) site, and an intronic splice enhancer (ISE) site,where when the SMO specifically binds to the complementary sequence, theexon adjacent to the intron-exon boundary is excluded from the resultingmRNA, where the exon is exon 3 of the mRNA encoding CypD, and where theSMO increases expression of a CypDΔ3 in the subject and treats thesubject afflicted with cancer. In one aspect, the SMO is an isolatednucleic acid sequence selected from the group consisting of SEQ ID NOs.858-918. In another aspect, the cancer is a liver cancer.

In yet another embodiment, the present invention comprises a method oftreating a subject afflicted with amyotrophic lateral sclerosis (ALS),the method comprising administering a splice modulating oligonucleotide(SMO) to the subject, where an effective amount of the SMO contacts acell so that the SMO specifically binds to a complementary sequence on apre-mRNA in at least one of the group consisting of an intron-exonsplice site, an exonic splice enhancer (ESE) site, and an intronicsplice enhancer (ISE) site, where when the SMO specifically binds to thecomplementary sequence, the exon adjacent to the intron-exon boundary isexcluded from the resulting mRNA, where the exon is exon 1 of the mRNAencoding CypD, and where the SMO increases expression of a CypDΔ1 in thesubject and treats the subject afflicted with ALS. In one aspect, theSMO is an isolated nucleic acid sequence selected from the groupconsisting of SEQ ID NOs. 814-857.

In another embodiment, the present invention comprises a method oftreating a subject afflicted with amyotrophic lateral sclerosis (ALS),the method comprising administering a splice modulating oligonucleotide(SMO) to the subject, where an effective amount of the SMO contacts acell so that the SMO specifically binds to a complementary sequence on apre-mRNA in at least one of the group consisting of an intron-exonsplice site, an exonic splice enhancer (ESE) site, and an intronicsplice enhancer (ISE) site, where when the SMO specifically binds to thecomplementary sequence, the exon adjacent to the intron-exon boundary isexcluded from the resulting mRNA, where the exon is exon 3 of the mRNAencoding CypD, and where the SMO increases expression of a CypDΔ3 in thesubject and treats the subject afflicted with ALS. In one aspect, theSMO is an isolated nucleic acid sequence selected from the groupconsisting of SEQ ID NOs. 858-918. In another aspect, the isolatednucleic acid selected from the group consisting of SEQ ID NOs. 1-1090.

In one embodiment, the present invention comprises a pharmaceuticalcomposition comprising a splice modulating oligonucleotide (SMO) thattargets a pre-mRNA that matures to a 5HT2CR, where the SMO is anisolated nucleic acid sequence selected from the group consisting of SEQID NOs. 1-56.

In still another embodiment, the present invention comprises apharmaceutical composition comprising a splice modulatingoligonucleotide (SMO) that targets a pre-mRNA that matures to a GluR,where the SMO is an isolated nucleic acid sequence selected from thegroup consisting of SEQ ID NOs. 57-526.

In yet another embodiment, the present invention comprises apharmaceutical composition comprising a splice modulatingoligonucleotide (SMO) that targets a pre-mRNA that matures to a OGA,where the SMO is an isolated nucleic acid sequence selected from thegroup consisting of SEQ ID NOs. 527-661.

In another embodiment, the present invention comprises a pharmaceuticalcomposition comprising a splice modulating oligonucleotide (SMO) thattargets a pre-mRNA that matures to a Aph1B, where the SMO is an isolatednucleic acid sequence selected from the group consisting of SEQ ID NOs.662-728.

In still another embodiment, the present invention a pharmaceuticalcomposition comprising a splice modulating oligonucleotide (SMO) thattargets a pre-mRNA that matures to a HER3, where the SMO is an isolatednucleic acid sequence selected from the group consisting of SEQ ID NOs.729-813.

In yet another embodiment, the present invention also comprises apharmaceutical composition comprising a splice modulatingoligonucleotide (SMO) that targets a pre-mRNA that matures to a CypD,where the SMO is an isolated nucleic acid sequence selected from thegroup consisting of SEQ ID NOs. 814-918.

In another embodiment, the present invention comprises a pharmaceuticalcomposition comprising a splice modulating oligonucleotide (SMO) thattargets a pre-mRNA that matures to a FOXM1, where the SMO is an isolatednucleic acid sequence selected from the group consisting of SEQ ID NOs.919-1090.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1, comprising FIG. 1A through FIG. 1I, is a series of imagesdepicting splice modulating oligonucleotide (SMO) mediated induction offull-length survival motor neuron protein (SMN) expression andconcomitant phenotypic improvement in spinal muscular atrophy (SMA)mice. FIG. 1A depicts a photomicrograph of brain section in anuninjected control. FIG. 1B depicts SMO fluorescent label broadlydistributed throughout brain regions 24 hours following bilateralintracerebroventricular (ICV) injection of the SMO. FIG. 1C is a highermagnification of the area within the box in FIG. 1B. FIG. 1D throughFIG. 1I depict results obtained from SMA mice (N=5), injected with 1 μgSMO (per ventricle) on postnatal day 1, 3, 5, 7, 10, and harvested onday 12, and compared with uninjected controls (N=9). FIG. 1D depicts theresults of real-time PCR of brain sections taken at the level ofhippocampus shows full-length SMN expression was increased in SMA micefollowing Icy injections of SMO. FIG. 1E and FIG. 1F depict Westernanalysis of brain sections taken at the level of hippocampus (FIG. 1E)and cervical spinal cord (FIG. 1F) showing SMN expression increases inSMA mice following ICV injections of SMO. FIG. 1G is a graph depictingSMN expression measured by Westerns as significantly increased in brainand spinal cord of SMO-treated SMA mice when measured as a percentage ofwild-type controls. FIG. 1H is a graph depicting body weight of SMA micewhich was significantly increased relative to un-injected controls atP12 following ICV injections of SMO (P<0.01). FIG. 1I is a graphdepicting SMO-treated SMA mice with significant improvement in rightingresponse at P12 compared to untreated controls. In total, all 5SMO-treated mice could right themselves from at least one side, whileonly 3 of 9 untreated mice could accomplish this task. However, motorfunction was not fully restored as most SMO-treated and untreated SMAmice could not right themselves from both sides.

FIG. 2 is a schematic illustration depicting alternative splicing at theflip-flop cassette exons of glutamate receptor (GluR) subunits of AMPAreceptors. Alternative splicing of mutually exclusive flip and flopexons of GluR1-4 leads to either flip exon-containing orflop-exon-containing transcripts. Co-skipping of both flip and flopexons results in out-of-frame transcripts that are truncated andunstable.

FIG. 3 depicts a ClustalW alignment of flip and flop exons of mouseGluR1-4. Dark shading indicates positions of complete identity, whilelighter shading shows divergence.

FIG. 4 is a schematic illustration of candidate SMOs evaluated forskipping GluR3 flip exon. All SMO-target pairs have favorablethermodynamic properties and are complementary to splice sites and/orESEs.

FIG. 5 is a schematic illustration of candidate SMOs evaluated forskipping GluR1 exon 14 (flip). All SMO-target pairs have favorablethermodynamic properties and are complementary to splice sites and/orESEs.

FIG. 6 is a graph depicting the relative expression of GluR1, GluR2,GluR3 and GluR4 flip and flop isoforms following ICV injections of SMOstargeting GluR1-flip and GluR3-flip isoforms.

FIG. 7 is a graph depicting the relative change in expression of GluR1,GluR2, Glur3 and GluR4 flip and flop isoforms following ICV injectionsof SMOs targeting all four GluR flip isoforms.

FIG. 8 is a graph depicting the effect of ICV administration of a SMOtargeting GluR-1 on seizure activity in mice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to splice modulating oligonucleotides(SMOs) affecting splicing of pre-mRNA expressed from various genes. Inone embodiment, the instant invention provides compositions and methodsfor correcting aberrant splicing of pre-mRNA that results in a defectiveprotein and consequently causes a disease or a disorder in a subject,wherein the subject is preferably human.

In another embodiment, the instant invention provides compositions andmethods for treating a human disease or disorder by modulating pre-mRNAsplicing of a nucleic acid even when that nucleic acid is not aberrantlyspliced in the pathogenesis of the disease or disorder being treated.

In one embodiment, the human disease or disorder is neurological. Inanother embodiment, the human disease is a cancer.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, and nucleic acidchemistry and hybridization are those well known and commonly employedin the art.

Standard techniques are used for nucleic acid and peptide synthesis. Thetechniques and procedures are generally performed according toconventional methods in the art and various general references (e.g.,Sambrook and Russell, 2001, Molecular Cloning, A Laboratory Approach,Cold Spring Harbor Press, Cold Spring Harbor, N.Y., and Ausubel et al.,2002, Current Protocols in Molecular Biology, John Wiley & Sons, NY),which are provided throughout this document.

The nomenclature used herein and the laboratory procedures used inanalytical chemistry and organic syntheses described below are thosewell known and commonly employed in the art. Standard techniques ormodifications thereof, are used for chemical syntheses and chemicalanalyses.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which it is used.

“Antisense” refers particularly to the nucleic acid sequence of thenon-coding strand of a double stranded DNA molecule encoding a protein,or to a sequence which is substantially homologous to the non-codingstrand. As defined herein, an antisense sequence is complementary to thesequence of a double stranded DNA molecule encoding a protein. It is notnecessary that the antisense sequence be complementary solely to thecoding portion of the coding strand of the DNA molecule. The antisensesequence may be complementary to regulatory sequences specified on thecoding strand of a DNA molecule encoding a protein, which regulatorysequences control expression of the coding sequences.

By the term “applicator,” as the term is used herein, is meant anydevice including, but not limited to, a hypodermic syringe, a pipette,and the like, for administering the compounds and compositions of theinvention.

“Complementary” as used herein refers to the broad concept of subunitsequence complementarity between two nucleic acids, e.g., two DNAmolecules. When a nucleotide position in both of the molecules isoccupied by nucleotides normally capable of base pairing with eachother, then the nucleic acids are considered to be complementary to eachother at this position. Thus, two nucleic acids are substantiallycomplementary to each other when at least about 50%, preferably at leastabout 60% and more preferably at least about 80% of correspondingpositions in each of the molecules are occupied by nucleotides whichnormally base pair with each other (e.g., A:T and G:C nucleotide pairs).

A “disease” is a state of health of subject wherein the subject cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe subject's health continues to deteriorate. In contrast, a “disorder”in an subject is a state of health in which the subject is able tomaintain homeostasis, but in which the subject's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe subject's state of health. In preferred embodiments, the subject isan animal. In more preferred embodiments, the subject is a mammal. Inmost preferred embodiments, the subject is a human.

A disease or disorder is “alleviated” if the severity of a symptom ofthe disease or disorder, or the frequency with which such a symptom isexperienced by a subject, or both, are reduced.

The terms “effective amount” and “pharmaceutically effective amount”refer to a nontoxic but sufficient amount of an agent to provide thedesired biological result. That result can be reduction and/oralleviation of the signs, symptoms, or causes of a disease or disorder,or any other desired alteration of a biological system. An appropriateeffective amount in any individual case may be determined by one ofordinary skill in the art using routine experimentation.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence.

The term “exonic regulatory elements” as used herein refers to sequencespresent on pre-mRNA that enhance or suppress splicing of an exon. Anexonic regulatory element that enhances splicing of an exon is an exonicsplicing enhancer (ESE). An exonic regulatory element that suppressessplicing of an exon is an exonic splicing suppressor (ESS). An intronicregulatory element that enhances splicing of an exon is an intronicsplicing enhancer (ISE). An intronic regulatory element that suppressessplicing of an exon is called an intronic splicing suppressor (ISS).

“Instructional material,” as that term is used herein, includes apublication, a recording, a diagram, or any other medium of expressionwhich can be used to communicate the usefulness of the compositionand/or compound of the invention in a kit. The instructional material ofthe kit may, for example, be affixed to a container that contains thecompound and/or composition of the invention or be shipped together witha container which contains the compound and/or composition.Alternatively, the instructional material may be shipped separately fromthe container with the intention that the recipient uses theinstructional material and the compound cooperatively. Delivery of theinstructional material may be, for example, by physical delivery of thepublication or other medium of expression communicating the usefulnessof the kit, or may alternatively be achieved by electronic transmission,for example by means of a computer, such as by electronic mail, ordownload from a website.

By “nucleic acid” is meant any nucleic acid, whether composed ofdeoxyribonucleosides or ribonucleosides, and whether composed ofphosphodiester linkages or modified linkages such as phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, phosphorothioate, methylphosphonate, phosphorodithioate,bridged phosphorothioate or sulfone linkages, and combinations of suchlinkages. The term nucleic acid also specifically includes nucleic acidscomposed of bases other than the five biologically occurring bases(adenine, guanine, thymine, cytosine and uracil). The term “nucleicacid” typically refers to large polynucleotides.

Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction.

The direction of 5′ to 3′ addition of nucleotides to nascent RNAtranscripts is referred to as the transcription direction. The DNAstrand having the same sequence as an mRNA is referred to as the “codingstrand”; sequences on the DNA strand which are located 5′ to a referencepoint on the DNA are referred to as “upstream sequences”; sequences onthe DNA strand which are 3′ to a reference point on the DNA are referredto as “downstream sequences.”

By “expression cassette” is meant a nucleic acid molecule comprising acoding sequence operably linked to promoter/regulatory sequencesnecessary for transcription and, optionally, translation of the codingsequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulator sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a n inducible manner.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced substantially only when aninducer which corresponds to the promoter is present.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds.Synthetic polypeptides can be synthesized, for example, using anautomated polypeptide synthesizer.

The term “protein” typically refers to large polypeptides.

The term “peptide” typically refers to short polypeptides.

Conventional notation is used herein to portray polypeptide sequences:the left-hand end of a polypeptide sequence is the amino-terminus; theright-hand end of a polypeptide sequence is the carboxyl-terminus.

A “polynucleotide” means a single strand or parallel and anti-parallelstrands of a nucleic acid. Thus, a polynucleotide may be either asingle-stranded or a double-stranded nucleic acid. In the context of thepresent invention, the following abbreviations for the commonlyoccurring nucleic acid bases are used. “A” refers to adenosine, “C”refers to cytidine, “G” refers to guanosine, “T” refers to thymidine,and “U” refers to uridine.

The term “oligonucleotide” typically refers to short polynucleotides,generally no greater than about 60 nucleotides. It will be understoodthat when a nucleotide sequence is represented by a DNA sequence (i.e.,A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) inwhich “U” replaces “T.”

The term “recombinant DNA” as used herein is defined as DNA produced byjoining pieces of DNA from different sources.

The term “recombinant polypeptide” as used herein is defined as apolypeptide produced by using recombinant DNA methods.

By the term “specifically binds,” as used herein, is meant a molecule,such as an antibody, which recognizes and binds to another molecule orfeature, but does not substantially recognize or bind other molecules orfeatures in a sample.

By the term “splice defect of a protein”, as used herein, is meant adefective protein resulting from a defect in the splicing of an RNAencoding a protein.

The term “treatment,” as used herein, refers to reversing, alleviating,delaying the onset of inhibiting the progress of, and/or preventing adisease or disorder, or one or more symptoms thereof, to which the termis applied in a subject. In some embodiments, treatment may be appliedafter one or more symptoms have developed. In other embodiments,treatment may be administered in the absence of symptoms. For example,treatment may be administered prior to symptoms (e.g., in light of ahistory of symptoms and/or one or more other susceptibility factors), orafter symptoms have resolved, for example to prevent or delay theirreoccurrence.

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual and partialnumbers within that range, for example, 1, 2, 3, 4, 5, 5.5 and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

The present invention encompasses a class of compounds known as splicemodulating oligonucleotides (SMOs) that modulate pre-mRNA splicing,thereby affecting expression and functionality of a specific protein ina cell. A SMO specifically binds to a complementary sequence on apre-mRNA at an exon or intron splice suppressor or splice enhancer site,or at an intron-exon splice site. When a SMO specifically binds to asplice enhancer site, or an intron-exon splice site, the adjacent exonis excluded from the resulting mRNA. In another embodiment, a SMOspecifically binds to a splice suppressor site or an intron-exon siteand the adjacent exon is included in the resulting mRNA. In anotherembodiment, a SMO specifically binds to a splice enhancer site or anintron-exon splice site and shifts the reading frame of the pre-mRNA sothat the resulting protein is a truncated. In some cases, the resultingprotein is a limited-function, or non-functional protein.

The location of an exonic or intronic splice enhancer or suppressormotif may be found anywhere within the exon and the flanking introns.Similarly, a SMO may either fully or partially overlap a predictedexonic or intronic splice enhancer or suppressor site in proximity to anintron-exon boundary and/or be complementary to the predicted 3′ or 5′splice sites.

I. Compositions: Splice Modulating Oligonucleotides

The present invention is directed, in part, to oligonucleotides referredto herein as splice modulating oligonucleotides (SMOs), suitable for usein modulating splicing of a target pre-mRNA both in vitro and in vivo.The present invention also includes a pharmaceutical compositioncomprising a SMO suitable for modulating splicing of a target pre-mRNAboth in vitro and in vivo. In vivo methodologies are useful for bothgeneral splice site modulatory purposes, as well as in therapeuticapplications in which modulating splicing of a target pre-mRNA isdesirable.

A. 5-Hydroxytryptamine (Serotonin) Receptor 2C (5-HT2CR)

The present invention provides SMOs based on the consensus sequence ofthe 5-HT2CR(HTR2c; MIM: 312861 GeneID: 3358), including upstream anddownstream nucleotides (Table 1). These SMOs are used according to themethods of the invention to modulate splicing of 5-HT2CR pre-mRNA. Inone embodiment, these SMOs are used according to the methods of theinvention to modulate splicing of 5-HT2CR pre-mRNA caused by a deletionof the 15q11-13 region of the 15^(th) chromosome that results in adeletion of the snoRNA HBII-52. In another embodiment, a SMO of theinvention functions to mimic the function of HBII-52. In anotherembodiment, a SMO of the invention functions to increase expression of afunctional 5-HT2CR transcript containing exon 5b.

In some embodiments, the invention includes a pharmaceutical compositionthat comprises a SMO that functions to modulate splicing of 5-HT2CRpre-mRNA. In other embodiments, the invention includes a pharmaceuticalcomposition that comprises a SMO that functions to modulate splicing of5-HT2CR pre-mRNA caused by a deletion of the 15q11-13 region of the15^(th) chromosome that results in a deletion of the snoRNA HBII-52. Inanother embodiment, the invention includes a pharmaceutical compositionthat comprises a SMO that functions to mimic the function of HBII-52. Instill another embodiment, the invention includes a pharmaceuticalcomposition that comprises a SMO that functions to increase expressionof a functional 5-HT2CR transcript containing exon 5b.

Table 1 depicts exemplary SMOs useful for modulating splicing of 5-HT2CRpre-mRNA in order to mimic the effect of HBII-52 snoRNA or increase theexpression or function of 5HT2CR containing exon 5b.

TABLE 1 SEQ 3′ to 5′ SMOs targeting the ID 5-HT2C pre-mRNA NO.5-HT2C sequence: 3′-CGAUACGAGUUAUCCUAAUGCAUA-5′ GCAUUAGGAUAACUCGUA  115 nucleotide (nt) SMO GGAUAACUCGUAUCG  2 AGGAUAACUCGUAUC  3UAGGAUAACUCGUAU  4 UUAGGAUAACUCGUA  5 AUUAGGAUAACUCGU  6 CAUUAGGAUAACUCG 7 GCAUUAGGAUAACUC  8 UGCAUUAGGAUAACU  9 AUGCAUUAGGAUAAC 10UAUGCAUUAGGAUAA 11 16 nt SMO AGGAUAACUCGUAUCG 12 UAGGAUAACUCGUAUC 13UUAGGAUAACUCGUAU 14 AUUAGGAUAACUCGUA 15 CAUUAGGAUAACUCGU 16GCAUUAGGAUAACUCG 17 UGCAUUAGGAUAACUC 18 AUGCAUUAGGAUAACU 19UAUGCAUUAGGAUAAC 20 17 nt SMO UAGGAUAACUCGUAUCG 21 UUAGGAUAACUCGUAUC 22AUUAGGAUAACUCGUAU 23 CAUUAGGAUAACUCGUA 24 GCAUUAGGAUAACUCGU 25UGCAUUAGGAUAACUCG 26 AUGCAUUAGGAUAACUC 27 UAUGCAUUAGGAUAACU 28 18 nt SMOGCAUUAGGAUAACUCGUA 29 UUAGGAUAACUCGUAUCG 30 AUUAGGAUAACUCGUAUC 31CAUUAGGAUAACUCGUAU 32 UGCAUUAGGAUAACUCGU 33 AUGCAUUAGGAUAACUCG 34UAUGCAUUAGGAUAACUC 35 19 nt SMO AUUAGGAUAACUCGUAUCG 36CAUUAGGAUAACUCGUAUC 37 GCAUUAGGAUAACUCGUAU 38 UGCAUUAGGAUAACUCGUA 39AUGCAUUAGGAUAACUCGU 40 UAUGCAUUAGGAUAACUCG 41 20 nt SMOCAUUAGGAUAACUCGUAUCG 42 GCAUUAGGAUAACUCGUAUC 43 UGCAUUAGGAUAACUCGUAU 44AUGCAUUAGGAUAACUCGUA 45 UAUGCAUUAGGAUAACUCGU 46 21 nt SMOGCAUUAGGAUAACUCGUAUCG 47 UGCAUUAGGAUAACUCGUAUC 48 AUGCAUUAGGAUAACUCGUAU49 UAUGCAUUAGGAUAACUCGUA 50 22 nt SMO UGCAUUAGGAUAACUCGUAUCG 51AUGCAUUAGGAUAACUCGUAUC 52 UAUGCAUUAGGAUAACUCGUAU 53 23 nt SMOAUGCAUUAGGAUAACUCGUAUCG 54 UAUGCAUUAGGAUAACUCGUAUC 55 24 nt SMOUAUGCAUUAGGAUAACUCGUAUCG 56

B. Glutamate Receptors

The present invention further provides SMOs based on the sequences ofthe flip and flop isoforms of GluR1 (GRIA1; MIM: 138248 GeneID: 2890),GluR2 (GRIA2;MIM: 138247 GeneID: 2891), GluR3 (GRIA3;MIM: 305915 GeneID:2892), and GluR4 (GRIA4;MIM: 138246 GeneID: 2893). These SMOs are usedaccording to the methods of the invention to modulate splicing of GluRpre-mRNA. In one embodiment, a SMO of the invention functions todecrease GluR flip isoform expression. In another embodiment, a SMO ofthe invention functions to decrease the GluR flip/flop isoform ratio. Inyet another embodiment, a SMO of the invention functions to increase theGluR flop isoform. In still another embodiment, a SMO of the inventionfunctions to increase the GluR flop isoforms. In various embodiments, aSMO of the invention functions to decrease both the GluR flip and GluRflop isoform expression.

In various embodiments, the invention includes a pharmaceuticalcomposition comprising a SMO of the invention, where the pharmaceuticalcomposition of the invention comprises a SMO that functions to decreasethe GluR flip isoform expression. In other embodiments, the inventionincludes a pharmaceutical composition comprising a SMO that decrease theGluR flip/flop isoform ratio of expression. In another embodiment, theinvention includes a pharmaceutical composition comprising a SMO thatfunctions to increase the GluR flop isoform expression. In yet anotherembodiment, the invention includes a pharmaceutical compositioncomprising a SMO of the invention that functions to decrease both theGluR flip and GluR flop isoform expression.

Table 2 depicts exemplary SMOs useful for modulating splicing of GluR3pre-mRNA in order to decrease GluR3-flip expression or increaseGluR3-flop expression in a cell.

TABLE 2 3′ to 5′ Splice modulating SEQ oligonucleotides directed  IDto GluR3-flip pre-mRNA NO. aaagggugcacuucUUGCGGACAU  57aagggugcacuucUUGCGGACAUU  58 agggugcacuucUUGCGGACAUUU  59gggugcacuucUUGCGGACAUUUG  60 ggugcacuucUUGCGGACAUUUGG  61gugcacuucUUGCGGACAUUUGGA  62 ugcacuucUUGCGGACAUUUGGAA  63gcacuucUUGCGGACAUUUGGAAC  64 cacuucUUGCGGACAUUUGGAACG  65acuucUUGCGGACAUUUGGAACGU  66 cuucUUGCGGACAUUUGGAACGUC  67uucUUGCGGACAUUUGGAACGUCA  68 ucUUGCGGACAUUUGGAACGUCAU  69cUUGCGGACAUUUGGAACGUCAUA  70 UUGCGGACAUUUGGAACGUCAUAA  71UGCGGACAUUUGGAACGUCAUAAC  72 GCGGACAUUUGGAACGUCAUAACU  73aaagggugcacuucUUGCGGACA  74 aagggugcacuucUUGCGGACAU  75agggugcacuucUUGCGGACAUU  76 gggugcacuucUUGCGGACAUUU  77ggugcacuucUUGCGGACAUUUG  78 gugcacuucUUGCGGACAUUUGG  79ugcacuucUUGCGGACAUUUGGA  80 gcacuucUUGCGGACAUUUGGAA  81cacuucUUGCGGACAUUUGGAAC  82 acuucUUGCGGACAUUUGGAACG  83cuucUUGCGGACAUUUGGAACGU  84 uucUUGCGGACAUUUGGAACGUC  85ucUUGCGGACAUUUGGAACGUCA  86 cUUGCGGACAUUUGGAACGUCAU  87UUGCGGACAUUUGGAACGUCAUA  88 UGCGGACAUUUGGAACGUCAUAA  89GCGGACAUUUGGAACGUCAUAAC  90 CGGACAUUUGGAACGUCAUAACU  91aaagggugcacuucUUGCGGAC  92 aagggugcacuucUUGCGGACA  93agggugcacuucUUGCGGACAU  94 gggugcacuucUUGCGGACAUU  95ggugcacuucUUGCGGACAUUU  96 gugcacuucUUGCGGACAUUUG  97ugcacuucUUGCGGACAUUUGG  98 gcacuucUUGCGGACAUUUGGA  99cacuucUUGCGGACAUUUGGAA 100 acuucUUGCGGACAUUUGGAAC 101cuucUUGCGGACAUUUGGAACG 102 uucUUGCGGACAUUUGGAACGU 103ucUUGCGGACAUUUGGAACGUC 104 cUUGCGGACAUUUGGAACGUCA 105UUGCGGACAUUUGGAACGUCAU 106 UGCGGACAUUUGGAACGUCAUA 107GCGGACAUUUGGAACGUCAUAA 108 CGGACAUUUGGAACGUCAUAAC 109GGACAUUUGGAACGUCAUAACU 110 aaagggugcacuucUUGCGGA 111aagggugcacuucUUGCGGAC 112 agggugcacuucUUGCGGACA 113gggugcacuucUUGCGGACAU 114 ggugcacuucUUGCGGACAUU 115gugcacuucUUGCGGACAUUU 116 ugcacuucUUGCGGACAUUUG 117gcacuucUUGCGGACAUUUGG 118 cacuucUUGCGGACAUUUGGA 119acuucUUGCGGACAUUUGGAA 120 cuucUUGCGGACAUUUGGAAC 121uucUUGCGGACAUUUGGAACG 122 ucUUGCGGACAUUUGGAACGU 123cUUGCGGACAUUUGGAACGUC 124 UUGCGGACAUUUGGAACGUCA 125UGCGGACAUUUGGAACGUCAU 126 GCGGACAUUUGGAACGUCAUA 127CGGACAUUUGGAACGUCAUAA 128 GGACAUUUGGAACGUCAUAAC 129GACAUUUGGAACGUCAUAACU 130 aaagggugcacuucUUGCGG 131 aagggugcacuucUUGCGGA132 agggugcacuucUUGCGGAC 133 gggugcacuucUUGCGGACA 134ggugcacuucUUGCGGACAU 135 gugcacuucUUGCGGACAUU 136 ugcacuucUUGCGGACAUUU137 gcacuucUUGCGGACAUUUG 138 cacuucUUGCGGACAUUUGG 139acuucUUGCGGACAUUUGGA 140 cuucUUGCGGACAUUUGGAA 141 uucUUGCGGACAUUUGGAAC142 ucUUGCGGACAUUUGGAACG 143 cUUGCGGACAUUUGGAACGU 144UUGCGGACAUUUGGAACGUC 145 UGCGGACAUUUGGAACGUCA 146 GCGGACAUUUGGAACGUCAU147 CGGACAUUUGGAACGUCAUA 148 GGACAUUUGGAACGUCAUAA 149aaagggugcacuucUUGCG 150 aagggugcacuucUUGCGG 151 agggugcacuucUUGCGGA 152gggugcacuucUUGCGGAC 153 ggugcacuucUUGCGGACA 154 gugcacuucUUGCGGACAU 155ugcacuucUUGCGGACAUU 156 gcacuucUUGCGGACAUUU 157 cacuucUUGCGGACAUUUG 158acuucUUGCGGACAUUUGG 159 cuucUUGCGGACAUUUGGA 160 uucUUGCGGACAUUUGGAA 161ucUUGCGGACAUUUGGAAC 162 cUUGCGGACAUUUGGAACG 163 UUGCGGACAUUUGGAACGU 164UGCGGACAUUUGGAACGUC 165 GCGGACAUUUGGAACGUCA 166 CGGACAUUUGGAACGUCAU 167GGACAUUUGGAACGUCAUA 168 aaagggugcacuucUUGC 169 aagggugcacuucUUGCG 170agggugcacuucUUGCGG 171 gggugcacuucUUGCGGA 172 ggugcacuucUUGCGGAC 173gugcacuucUUGCGGACA 174 ugcacuucUUGCGGACAU 175 gcacuucUUGCGGACAUU 176cacuucUUGCGGACAUUU 177 acuucUUGCGGACAUUUG 178 cuucUUGCGGACAUUUGG 179uucUUGCGGACAUUUGGA 180 ucUUGCGGACAUUUGGAA 181 cUUGCGGACAUUUGGAAC 182UUGCGGACAUUUGGAACG 183 UGCGGACAUUUGGAACGU 184 GCGGACAUUUGGAACGUC 185CGGACAUUUGGAACGUCA 186 GGACAUUUGGAACGUCAU 187

Table 3 depicts exemplary SMOs for modulating splicing of GluR1 pre-mRNAin order to decrease GluR1-flip expression or increase GluR1-flopexpression in a cell.

TABLE 3 3′ to 5′ Splice modulating SEQ  oligonucleotides directed  IDto GluR1-flip pre-mRNA NO. caacuucUCCAGGGCAUUUGGAUC 188aacuucUCCAGGGCAUUUGGAUCG 189 acuucUCCAGGGCAUUUGGAUCGC 190cuucUCCAGGGCAUUUGGAUCGCC 191 uucUCCAGGGCAUUUGGAUCGCCA 192ucUCCAGGGCAUUUGGAUCGCCAA 193 cUCCAGGGCAUUUGGAUCGCCAAA 194UCCAGGGCAUUUGGAUCGCCAAAA 195 caacuucUCCAGGGCAUUUGGAU 196aacuucUCCAGGGCAUUUGGAUC 197 acuucUCCAGGGCAUUUGGAUCG 198cuucUCCAGGGCAUUUGGAUCGC 199 uucUCCAGGGCAUUUGGAUCGCC 200ucUCCAGGGCAUUUGGAUCGCCA 201 cUCCAGGGCAUUUGGAUCGCCAA 202UCCAGGGCAUUUGGAUCGCCAAA 203 CCAGGGCAUUUGGAUCGCCAAAA 204caacuucUCCAGGGCAUUUGGA 205 aacuucUCCAGGGCAUUUGGAU 206acuucUCCAGGGCAUUUGGAUC 207 cuucUCCAGGGCAUUUGGAUCG 208uucUCCAGGGCAUUUGGAUCGC 209 ucUCCAGGGCAUUUGGAUCGCC 210cUCCAGGGCAUUUGGAUCGCCA 211 UCCAGGGCAUUUGGAUCGCCAA 212CCAGGGCAUUUGGAUCGCCAAA 213 caacuucUCCAGGGCAUUUGG 214aacuucUCCAGGGCAUUUGGA 215 acuucUCCAGGGCAUUUGGAU 216cuucUCCAGGGCAUUUGGAUC 217 uucUCCAGGGCAUUUGGAUCG 218ucUCCAGGGCAUUUGGAUCGC 219 cUCCAGGGCAUUUGGAUCGCC 220UCCAGGGCAUUUGGAUCGCCA 221 CCAGGGCAUUUGGAUCGCCAA 222 caacuucUCCAGGGCAUUUG223 aacuucUCCAGGGCAUUUGG 224 acuucUCCAGGGCAUUUGGA 225cuucUCCAGGGCAUUUGGAU 226 uucUCCAGGGCAUUUGGAUC 227 ucUCCAGGGCAUUUGGAUCG228 cUCCAGGGCAUUUGGAUCGC 229 UCCAGGGCAUUUGGAUCGCC 230CCAGGGCAUUUGGAUCGCCA 231 caacuucUCCAGGGCAUUU 232 aacuucUCCAGGGCAUUUG 233acuucUCCAGGGCAUUUGG 234 cuucUCCAGGGCAUUUGGA 235 uucUCCAGGGCAUWGGAU 236ucUCCAGGGCAUUUGGAUC 237 cUCCAGGGCAUUUGGAUCG 238 UCCAGGGCAUUUGGAUCGC 239CCAGGGCAUUUGGAUCGCC 240 caacuucUCCAGGGCAUU 241 aacuucUCCAGGGCAUUU 242acuucUCCAGGGCAUUUG 243 cuucUCCAGGGCAUUUGG 244 uucUCCAGGGCAUUUGGA 245ucUCCAGGGCAUUUGGAU 246 cUCCAGGGCAUUUGGAUC 247 UCCAGGGCAUUUGGAUCG 248CCAGGGCAUUUGGAUCGC 249 ACCUUCGUUCCUGAGGCCUUCAUU 250CCUUCGUUCCUGAGGCCUUCAUUC 251 CUUCGUUCCUGAGGCCUUCAUUCc 252UUCGUUCCUGAGGCCUUCAUUCca 253 UCGUUCCUGAGGCCUUCAUUCcag 254CGUUCCUGAGGCCUUCAUUCcagu 255 GUUCCUGAGGCCUUCAUUCcaguc 256UUCCUGAGGCCUUCAUUCcaguca 257 CCUUCGUUCCUGAGGCCUUCAUU 258CUUCGUUCCUGAGGCCUUCAUUC 259 UUCGUUCCUGAGGCCUUCAUUCc 260UCGUUCCUGAGGCCUUCAUUCca 261 CGUUCCUGAGGCCUUCAUUCcag 262GUUCCUGAGGCCUUCAUUCcagu 263 UUCCUGAGGCCUUCAUUCcaguc 264UCCUGAGGCCUUCAUUCcaguca 265 CUUCGUUCCUGAGGCCUUCAUU 266UUCGUUCCUGAGGCCUUCAUUC 267 UCGUUCCUGAGGCCUUCAUUCc 268CGUUCCUGAGGCCUUCAUUCca 269 GUUCCUGAGGCCUUCAUUCcag 270UUCCUGAGGCCUUCAUUCcagu 271 UCCUGAGGCCUUCAUUCcaguc 272CCUGAGGCCUUCAUUCcaguca 273 UUCGUUCCUGAGGCCUUCAUU 274UCGUUCCUGAGGCCUUCAUUC 275 CGUUCCUGAGGCCUUCAUUCc 276GUUCCUGAGGCCUUCAUUCca 277 UUCCUGAGGCCUUCAUUCcag 278UCCUGAGGCCUUCAUUCcagu 279 CCUGAGGCCUUCAUUCcaguc 280CUGAGGCCUUCAUUCcaguca 281 UCGUUCCUGAGGCCUUCAUU 282 CGUUCCUGAGGCCUUCAUUC283 GUUCCUGAGGCCUUCAUUCc 284 UUCCUGAGGCCUUCAUUCca 285UCCUGAGGCCUUCAUUCcag 286 CCUGAGGCCUUCAUUCcagu 287 CUGAGGCCUUCAUUCcaguc288 UGAGGCCUUCAUUCcaguca 289 CGUUCCUGAGGCCUUCAUU 290 GUUCCUGAGGCCUUCAUUC291 UUCCUGAGGCCUUCAUUCc 292 UCCUGAGGCCUUCAUUCca 293 CCUGAGGCCUUCAUUCcag294 CUGAGGCCUUCAUUCcagu 295 UGAGGCCUUCAUUCcaguc 296 GAGGCCUUCAUUCcaguca297 GUUCCUGAGGCCUUCAUU 298 UUCCUGAGGCCUUCAUUC 299 UCCUGAGGCCUUCAUUCc 300CCUGAGGCCUUCAUUCca 301 CUGAGGCCUUCAUUCcag 302 UGAGGCCUUCAUUCcagu 303GAGGCCUUCAUUCcaguc 304 AGGCCUUCAUUCcaguca 305

Table 4 depicts exemplary SMOs for modulating splicing of all GluRsubtypes, including GluR1, GluR2, GluR3, and GluR4 pre-mRNA in order todecrease GluR1-4-flip expression or increase GluR1-4-flop expression ina cell.

TABLE 4 SEQ 3′ to 5′ SMOs targeting GluR1, ID GluR2, GluR3, and GluR4NO. UUCCGCAGAUUCUGUUCGACUUUU 306 UUCCGCAGAUUCUGUUCGACUUU 307UCCGCAGAUUCUGUUCGACUUUU 308 UUCCGCAGAUUCUGUUCGACUU 309UCCGCAGAUUCUGUUCGACUUU 310 CCGCAGAUUCUGUUCGACUUUU 311UUCCGCAGAUUCUGUUCGACU 312 UCCGCAGAUUCUGUUCGACUU 313CCGCAGAUUCUGUUCGACUUU 314 CGCAGAUUCUGUUCGACUUUU 315 UUCCGCAGAUUCUGUUCGAC316 UCCGCAGAUUCUGUUCGACU 317 CCGCAGAUUCUGUUCGACUU 318CGCAGAUUCUGUUCGACUUU 319 GCAGAUUCUGUUCGACUUUU 320 UUCCGCAGAUUCUGUUCGA321 UCCGCAGAUUCUGUUCGAC 322 CCGCAGAUUCUGUUCGACU 323 CGCAGAUUCUGUUCGACUU324 GCAGAUUCUGUUCGACUUU 325 CAGAUUCUGUUCGACUUUU 326 UUCCGCAGAUUCUGUUCG327 UCCGCAGAUUCUGUUCGA 328 CCGCAGAUUCUGUUCGAC 329 CGCAGAUUCUGUUCGACU 330GCAGAUUCUGUUCGACUU 331 CAGAUUCUGUUCGACUUU 332 AGAUUCUGUUCGACUUUU 333UUGUUCCGUAGAAUCUGUUCGACU 334 UGUUCCGUAGAAUCUGUUCGACUU 335GUUCCGUAGAAUCUGUUCGACUUU 336 UUCCGUAGAAUCUGUUCGACUUUU 337UUGUUCCGUAGAAUCUGUUCGAC 338 UGUUCCGUAGAAUCUGUUCGACU 339GUUCCGUAGAAUCUGUUCGACUU 340 UUCCGUAGAAUCUGUUCGACUUU 341UCCGUAGAAUCUGUUCGACUUUU 342 UUGUUCCGUAGAAUCUGUUCGA 343UGUUCCGUAGAAUCUGUUCGAC 344 GUUCCGUAGAAUCUGUUCGACU 345UUCCGUAGAAUCUGUUCGACUU 346 UCCGUAGAAUCUGUUCGACUUU 347CCGUAGAAUCUGUUCGACUUUU 348 UUGUUCCGUAGAAUCUGUUCG 349UGUUCCGUAGAAUCUGUUCGA 350 GUUCCGUAGAAUCUGUUCGAC 351UUCCGUAGAAUCUGUUCGACU 352 UCCGUAGAAUCUGUUCGACUU 353CCGUAGAAUCUGUUCGACUUU 354 CGUAGAAUCUGUUCGACUUUU 355 UUGUUCCGUAGAAUCUGUUC356 UGUUCCGUAGAAUCUGUUCG 357 GUUCCGUAGAAUCUGUUCGA 358UUCCGUAGAAUCUGUUCGAC 359 UCCGUAGAAUCUGUUCGACU 360 CCGUAGAAUCUGUUCGACUU361 UUCCGUAGAAUCUGUUCGA 362 UCCGUAGAAUCUGUUCGAC 363 CCGUAGAAUCUGUUCGACU364 CGUAGAAUCUGUUCGACUU 365 UUCCGUAGAAUCUGUUCG 366 UCCGUAGAAUCUGUUCGA367 CCGUAGAAUCUGUUCGAC 368 CGUAGAAUCUGUUCGACU 369 ACUUUUCGUUUACCACCAUGCU370 ACUUUUCGUUUACCACCAUGC 371 CUUUUCGUUUACCACCAUGCU 372CUUUUCGUUUACCACCAUGC 373 CUUUUCGUUUACCACCAUGC 374 UUUUCGUUUACCACCAUGCU375 UUUGAGUCACUUGUUCCGUAGAAU 376 UUUGAGUCACUUGUUCCGUAGAA  377UUGAGUCACUUGUUCCGUAGAAU  378 UUUGAGUCACUUGUUCCGUAGA 379UUGAGUCACUUGUUCCGUAGAA 380 UGAGUCACUUGUUCCGUAGAAU 381UUUGAGUCACUUGUUCCGUAG 382 UUGAGUCACUUGUUCCGUAGA 383UGAGUCACUUGUUCCGUAGAA 384 GAGUCACUUGUUCCGUAGAAU 385 UUUGAGUCACUUGUUCCGUA386 UUGAGUCACUUGUUCCGUAG 387 UGAGUCACUUGUUCCGUAGA 388GAGUCACUUGUUCCGUAGAA 389 AGUCACUUGUUCCGUAGAAU 390 UUUGAGUCACUUGUUCCGU391 UUGAGUCACUUGUUCCGUA 392 UGAGUCACUUGUUCCGUAG 393 GAGUCACUUGUUCCGUAGA394 AGUCACUUGUUCCGUAGAA 395 GUCACUUGUUCCGUAGAAU 396 UUGAGUCACUUGUUCCGU397 UGAGUCACUUGUUCCGUA 398 GAGUCACUUGUUCCGUAG 399 AGUCACUUGUUCCGUAGA 400

Table 5 depicts exemplary SMOs for modulating splicing of GluR2 pre-mRNAin order to decrease GluR2-flip expression or increase GluR2-flopexpression in a cell.

TABLE 5 3′ to 5′ Splice modulating SEQ oligonucleotidesdirected IDto flip GluR2 NO. gcacuucUUGGGGUCAUUUAGAAC 401 cacuucUUGGGGUCAUUUAGAACG402 acuucUUGGGGUCAUUUAGAACGU 403 cuucUUGGGGUCAUUUAGAACGUC 404uucUUGGGGUCAUUUAGAACGUCA 405 ucUUGGGGUCAUUUAGAACGUCAU 406cUUGGGGUCAUUUAGAACGUCAUA 407 UUGGGGUCAUUUAGAACGUCAUAA 408UGGGGUCAUUUAGAACGUCAUAAC 409 gcacuucUUGGGGUCAUUUAGAA 410cacuucUUGGGGUCAUUUAGAAC 411 acuucUUGGGGUCAUUUAGAACG 412cuucUUGGGGUCAUUUAGAACGU 413 uucUUGGGGUCAUUUAGAACGUC 414ucUUGGGGUCAUUUAGAACGUCA 415 cUUGGGGUCAUUUAGAACGUCAU 416UUGGGGUCAUUUAGAACGUCAUA 417 UGGGGUCAUUUAGAACGUCAUAA 418gcacuucUUGGGGUCAUUUAGA 419 cacuucUUGGGGUCAUUUAGAA 420acuucUUGGGGUCAUUUAGAAC 421 cuucUUGGGGUCAUUUAGAACG 422uucUUGGGGUCAUUUAGAACGU 423 ucUUGGGGUCAUUUAGAACGUC 424cUUGGGGUCAUUUAGAACGUCA 425 UUGGGGUCAUUUAGAACGUCAU 426UGGGGUCAUUUAGAACGUCAUA 427 gcacuucUUGGGGUCAUUUAG 428cacuucUUGGGGUCAUUUAGA 429 acuucUUGGGGUCAUUUAGAA 430cuucUUGGGGUCAUUUAGAAC 431 uucUUGGGGUCAUUUAGAACG 432ucUUGGGGUCAUUUAGAACGU 433 cUUGGGGUCAUUUAGAACGUC 434UUGGGGUCAUUUAGAACGUCA 435 UGGGGUCAUUUAGAACGUCAU 436 gcacuucUUGGGGUCAUUUA437 cacuucUUGGGGUCAUUUAG 438 acuucUUGGGGUCAUUUAGA 439cuucUUGGGGUCAUUUAGAA 440 uucUUGGGGUCAUUUAGAAC 441 ucUUGGGGUCAUUUAGAACG442 cUUGGGGUCAUUUAGAACGU 443 UUGGGGUCAUUUAGAACGUC 444UGGGGUCAUUUAGAACGUCA 445 gcacuucUUGGGGUCAUUU 446 cacuucUUGGGGUCAUUUA 447acuucUUGGGGUCAUUUAG 448 cuucUUGGGGUCAUUUAGA 449 uucUUGGGGUCAUUUAGAA 450ucUUGGGGUCAUUUAGAAC 451 cUUGGGGUCAUUUAGAACG 452 UUGGGGUCAUUUAGAACGU 453UGGGGUCAUUUAGAACGUC 454 gcacuucUUGGGGUCAUU 455 cacuucUUGGGGUCAUUU 456acuucUUGGGGUCAUUUA 457 cuucUUGGGGUCAUUUAG 458 uucUUGGGGUCAUUUAGA 459ucUUGGGGUCAUUUAGAA 460 cUUGGGGUCAUUUAGAAC 461 UUGGGGUCAUUUAGAACG 462UGGGGUCAUUUAGAACGU 463

Table 6 depicts exemplary SMOs for modulating splicing of GluR4 pre-mRNAin order to decrease GluR4-flip expression or increase GluR4-flopexpression in a cell.

TABLE 6 3′ to 5′ Splice modulating SEQ oligonucleotides directed IDto all flip GluR4 NO. gcacuucUUGAGGACAUUUGGAAC 464cacuucUUGAGGUCAUUUGGAACG 465 acuucUUGAGGUCAUUUGGAACGG 466cuucUUGAGGUCAUUUGGAACGGC 467 uucUUGAGGUCAUUUGGAACGGCA 468ucUUGAGGUCAUUUGGAACGGCAA 469 cUUGAGGUCAUUUGGAACGGCAAA 470UUGAGGUCAUUUGGAACGGCAAAA 471 UGAGGUCAUUUGGAACGGCAAAAC 472gcacuucUUGAGGACAUUUGGAA 473 cacuucUUGAGGUCAUUUGGAAC 474acuucUUGAGGUCAUUUGGAACG 475 cuucUUGAGGUCAUUUGGAACGG 476uucUUGAGGUCAUUUGGAACGGC 477 ucUUGAGGUCAUUUGGAACGGCA 478cUUGAGGUCAUUUGGAACGGCAA 479 UUGAGGUCAUUUGGAACGGCAAA 480UGAGGUCAUUUGGAACGGCAAAA 481 gcacuucUUGAGGACAUUUGGA 482cacuucUUGAGGUCAUUUGGAA 483 acuucUUGAGGUCAUUUGGAAC 484cuucUUGAGGUCAUUUGGAACG 485 uucUUGAGGUCAUUUGGAACGG 486ucUUGAGGUCAUUUGGAACGGC 487 cUUGAGGUCAUUUGGAACGGCA 488UUGAGGUCAUUUGGAACGGCAA 489 UGAGGUCAUUUGGAACGGCAAA 490gcacuucUUGAGGACAUUUGG 491 cacuucUUGAGGUCAUUUGGA 492acuucUUGAGGUCAUUUGGAA 493 cuucUUGAGGUCAUUUGGAAC 494uucUUGAGGUCAUUUGGAACG 495 ucUUGAGGUCAUUUGGAACGG 496cUUGAGGUCAUUUGGAACGGC 497 UUGAGGUCAUUUGGAACGGCA 498UGAGGUCAUUUGGAACGGCAA 499 gcacuucUUGAGGACAUUUG 500 cacuucUUGAGGUCAUUUGG501 acuucUUGAGGUCAUUUGGA 502 cuucUUGAGGUCAUUUGGAA 503uucUUGAGGUCAUUUGGAAC 504 ucUUGAGGUCAUUUGGAACG 505 cUUGAGGUCAUUUGGAACGG506 UUGAGGUCAUUUGGAACGGC 507 UGAGGUCAUUUGGAACGGCA 508gcacuucUUGAGGACAUUU 509 cacuucUUGAGGUCAUUUG 510 acuucUUGAGGUCAUUUGG 511cuucUUGAGGUCAUUUGGA 512 uucUUGAGGUCAUUUGGAA 513 ucUUGAGGUCAUUUGGAAC 514cUUGAGGUCAUUUGGAACG 515 UUGAGGUCAUUUGGAACGG 516 UGAGGUCAUUUGGAACGGC 517gcacuucUUGAGGACAUU 518 cacuucUUGAGGUCAUUU 519 acuucUUGAGGUCAUUUG 520cuucUUGAGGUCAUUUGG 521 uucUUGAGGUCAUUUGGA 522 ucUUGAGGUCAUUUGGAA 523cUUGAGGUCAUUUGGAAC 524 UUGAGGUCAUUUGGAACG 525 UGAGGUCAUUUGGAACGG 526

C. O-GlcNAcase (OGA)

The present invention further provides SMOs based on the sequences ofOGA (MGEA5; MIM: 604039; GeneID: 10724). These SMOs are used accordingto the methods of the invention to modulate splicing of OGA pre-mRNA. Inone embodiment, a SMO of the invention functions to decrease OGAexpression or function. In another embodiment, the invention includes apharmaceutical composition comprising a SMO of the invention, where thepharmaceutical composition of the invention comprises a SMO thatfunctions to decrease the OGA expression or function. In one aspect, analternative splice variant of OGA with reduced catalytic activitycomprises OGA 10t, a read-through variant which results in 15 aminoacids being added from intron 10. In another aspect, an alternativesplice variant of OGA with reduced catalytic activity comprises OGAΔ8wherein exon 8 of the OGA gene is excluded.

Table7 depicts exemplary SMOs for modulating splicing of exon 8 of OGApre-mRNA in order to produce an OGA protein with lower enzymaticactivity in a cell.

TABLE 7 3′ to 5′ Splice modulating SEQ oligonucleotides targeting IDExon 8 of OGA NO. gucGACUGUCACUUCUGUCAUGAC 527 ucGACUGUCACUUCUGUCAUGACA528 cGACUGUCACUUCUGUCAUGACAU 529 UCUUUUACUUCCGUCACUGCUUCU 530CACUGCUUCUGUAACUUUGACUAC 531 ACUGCUUCUGUAACUUUGACUACA 532gucGACUGUCACUUCUGUCAUGA 533 ucGACUGUCACUUCUGUCAUGAC 534cGACUGUCACUUCUGUCAUGACA 535 UCUUUUACUUCCGUCACUGCUUC 536CUUUUACUUCCGUCACUGCUUCU 537 CACUGCUUCUGUAACUUUGACUA 538ACUGCUUCUGUAACUUUGACUAC 539 CUGCUUCUGUAACUUUGACUACA 540GGAGUAGUUAUGUCGUcacucaa 541 gucGACUGUCACUUCUGUCAUG 542ucGACUGUCACUUCUGUCAUGA 543 cGACUGUCACUUCUGUCAUGAC 544UCUUUUACUUCCGUCACUGCUU 545 CUUUUACUUCCGUCACUGCUUC 546UUUUACUUCCGUCACUGCUUCU 547 CACUGCUUCUGUAACUUUGACU 548ACUGCUUCUGUAACUUUGACUA 549 CUGCUUCUGUAACUUUGACUAC 550UGCUUCUGUAACUUUGACUACA 551 GGAGUAGUUAUGUCGUcacuca 552GAGUAGUUAUGUCGUcacucaa 553 gucGACUGUCACUUCUGUCAU 554ucGACUGUCACUUCUGUCAUG 555 cGACUGUCACUUCUGUCAUGA 556UCUUUUACUUCCGUCACUGCU 557 CUUUUACUUCCGUCACUGCUU 558UUUUACUUCCGUCACUGCUUC 559 UUUACUUCCGUCACUGCUUCU 560CACUGCUUCUGUAACUUUGAC 561 ACUGCUUCUGUAACUUUGACU 562CUGCUUCUGUAACUUUGACUA 563 UGCUUCUGUAACUUUGACUAC 564GCUUCUGUAACUUUGACUACA 565 GGAGUAGUUAUGUCGUcacuc 566GAGUAGUUAUGUCGUcacuca 567 AGUAGUUAUGUCGUcacucaa 568 gucGACUGUCACUUCUGUCA569 ucGACUGUCACUUCUGUCAU 570 cGACUGUCACUUCUGUCAUG 571UCUUUUACUUCCGUCACUGC 572 CUUUUACUUCCGUCACUGCU 573 UUUUACUUCCGUCACUGCUU574 UUUACUUCCGUCACUGCUUC 575 UUACUUCCGUCACUGCUUCU 576CACUGCUUCUGUAACUUUGA 577 ACUGCUUCUGUAACUUUGAC 578 CUGCUUCUGUAACUUUGACU579 UGCUUCUGUAACUUUGACUA 580 GCUUCUGUAACUUUGACUAC 581CUUCUGUAACUUUGACUACA 582 GGAGUAGUUAUGUCGUcacu 583 GAGUAGUUAUGUCGUcacuc584 AGUAGUUAUGUCGUcacuca 585 GUAGUUAUGUCGUcacucaa 586gucGACUGUCACUUCUGUC 587 ucGACUGUCACUUCUGUCA 588 cGACUGUCACUUCUGUCAU 589UCUUUUACUUCCGUCACUG 590 CUUUUACUUCCGUCACUGC 591 UUUUACUUCCGUCACUGCU 592UUUACUUCCGUCACUGCUU 593 UUACUUCCGUCACUGCUUC 594 UACUUCCGUCACUGCUUCU 595GGAGUAGUUAUGUCGUcac 596 GAGUAGUUAUGUCGUcacu 597 AGUAGUUAUGUCGUcacuc 598GUAGUUAUGUCGUcacuca 599 UAGUUAUGUCGUcacucaa 600 agugucGACUGUCACUUC 601gucGACUGUCACUUCUGU 602 ucGACUGUCACUUCUGUC 603 cGACUGUCACUUCUGUCA 604ACUUCCGUCACUGCUUCU 605 GGAGUAGUUAUGUCGUca 606 GAGUAGUUAUGUCGUcac 607AGUAGUUAUGUCGUcacu 608 GUAGUUAUGUCGUcacuc 609 UAGUUAUGUCGUcacuca 610AGUUAUGUCGUcacucaa 611

Table 8 depicts exemplary SMOs for modulating splicing of exon 10 of OGApre-mRNA in order to produce an OGA protein with lower enzymaticactivity in a cell.

TABLE 8 3′ to 5′ Splice modulating SEQ oligonucleotides directed IDexon 10 of OGA NO. UUUAGAAAACAUGUCACCAAUCca 612 UUAGAAAACAUGUCACCAAUCcau613 UAGAAAACAUGUCACCAAUCcauc 614 AGAAAACAUGUCACCAAUCcaucc 615GAAAACAUGUCACCAAUCcaucca 616 UUAGAAAACAUGUCACCAAUCca 617UAGAAAACAUGUCACCAAUCcau 618 AGAAAACAUGUCACCAAUCcauc 619GAAAACAUGUCACCAAUCcaucc 620 AAAACAUGUCACCAAUCcaucca 621UAGAAAACAUGUCACCAAUCca 622 AGAAAACAUGUCACCAAUCcau 623GAAAACAUGUCACCAAUCcauc 624 AAAACAUGUCACCAAUCcaucc 625AAACAUGUCACCAAUCcaucca 626 GAUACCACUUUAGAAAACAUGU 627AGAAAACAUGUCACCAAUCca 628 GAAAACAUGUCACCAAUCcau 629AAAACAUGUCACCAAUCcauc 630 AAACAUGUCACCAAUCcaucc 631AACAUGUCACCAAUCcaucca 632 GAUACCACUUUAGAAAACAUG 633AUACCACUUUAGAAAACAUGU 634 GAAAACAUGUCACCAAUCca 635 AAAACAUGUCACCAAUCcau636 AAACAUGUCACCAAUCcauc 637 AACAUGUCACCAAUCcaucc 638ACAUGUCACCAAUCcaucca 639 GAUACCACUUUAGAAAACAU 640 AUACCACUUUAGAAAACAUG641 UACCACUUUAGAAAACAUGU 642 AAAACAUGUCACCAAUCca 643 AAACAUGUCACCAAUCcau644 AACAUGUCACCAAUCcauc 645 ACAUGUCACCAAUCcaucc 646 CAUGUCACCAAUCcaucca647 GAUACCACUUUAGAAAACA 648 AUACCACUUUAGAAAACAU 649 UACCACUUUAGAAAACAUG650 ACCACUUUAGAAAACAUGU 651 AAACAUGUCACCAAUCca 652 AACAUGUCACCAAUCcau653 ACAUGUCACCAAUCcauc 654 CAUGUCACCAAUCcaucc 655 AUGUCACCAAUCcaucca 656GAUACCACUUUAGAAAAC 657 AUACCACUUUAGAAAACA 658 UACCACUUUAGAAAACAU 659ACCACUUUAGAAAACAUG 660 CCACUUUAGAAAACAUGU 661

D. Aph1B

The present invention further provides SMOs based on the sequences ofAph1B (APH1B; MIM: 607630; GeneID: 83464). These SMOs are used accordingto the methods of the invention to modulate splicing of Aph1B pre-mRNA.In one embodiment, a SMO of the invention functions to decrease Aph1Bexpression or function. In another embodiment, the invention includes apharmaceutical composition comprising a SMO of the invention, where thepharmaceutical composition of the invention comprises a SMO thatfunctions to decrease the Aph1B expression or function. In one aspect,the SMO contacts an Aph1B pre-mRNA and modulates the splicing of theAph1B pre-mRNA such that “in-frame” exon 4 is skipped, resulting inAph1BΔ4, a non-functional protein.

Table 9 depicts exemplary SMOs for modulating splicing of Alph1Bpre-mRNA in order to produce a non-functional protein with lowerenzymatic activity in a cell.

TABLE 9 3′ to 5′ Splice modulating SEQ oligonucleotides directed  IDto exon 4 of Aph 1B NO. aaaagaaggacaaaucAAAGAC 662aaagaaggacaaaucAAAGACC 663 aagaaggacaaaucAAAGACC 664aagaaggacaaaucAAAGAC 665 agaaggacaaaucAAAGACC 666 agaaggacaaaucAAAGAC667 gaaggacaaaucAAAGACC 668 aaggacaaaucAAAGACC 669 GAAACCUUAGUACUCACCUCA670 AAACCUUAGUACUCACCUCA 671 AACCUUAGUACUCACCUCA 672 AACCUUAGUACUCACCUC673 GAAACCUUAGUACUCACCUC 674 AACCUUAGUACUCACCUCAU 675ACCUUAGUACUCACCUCAUA 676 CCUUAGUACUCACCUCAUAA 677 GAAACCUUAGUACUCACCU678 AAACCUUAGUACUCACCUC 679 ACCUUAGUACUCACCUCAU 680 CCUUAGUACUCACCUCAUA681 ACCUUAGUACUCACCUCA 682 CCUUAGUACUCACCUCAU 683 GGUCCGUGUCACCCGUAAGU684 GUCCGUGUCACCCGUAAGUA 685 UCCGUGUCACCCGUAAGUAC 686CCGUGUCACCCGUAAGUACC 687 GGUCCGUGUCACCCGUAAG 688 GUCCGUGUCACCCGUAAGU 689UCCGUGUCACCCGUAAGUA 690 CCGUGUCACCCGUAAGUAC 691 CGUGUCACCCGUAAGUACC 692GGUCCGUGUCACCCGUAA 693 GUCCGUGUCACCCGUAAG 694 UCCGUGUCACCCGUAAGU 695CCGUGUCACCCGUAAGUA 696 CGUGUCACCCGUAAGUAC 697 GUGUCACCCGUAAGUACC 698AUAAGUCcauacacagaguauc 699 UAAGUCcauacacagaguaucg 700AAGUCcauacacagaguaucga 701 AGUCcauacacagaguaucgac 702GUCcauacacagaguaucgaca 703 UCcauacacagaguaucgacag 704Ccauacacagaguaucgacagu 705 AUAAGUCcauacacagaguau 706UAAGUCcauacacagaguauc 707 AAGUCcauacacagaguaucg 708AGUCcauacacagaguaucga 709 GUCcauacacagaguaucgac 710UCcauacacagaguaucgaca 711 AUAAGUCcauacacagagua 712 UAAGUCcauacacagaguau713 AAGUCcauacacagaguauc 714 AGUCcauacacagaguaucg 715GUCcauacacagaguaucga 716 UCcauacacagaguaucgac 717 Ccauacacagaguaucgaca718 AUAAGUCcauacacagagu 719 AAGUCcauacacagaguau 720 AGUCcauacacagaguauc721 GUCcauacacagaguaucg 722 UCcauacacagaguaucga 723 Ccauacacagaguaucgac724 GUCcauacacagaguauc 725 UCcauacacagaguaucg 726 Ccauacacagaguaucga 727UAAGUCcauacac 728

E. HER3

The present invention further provides SMOs based on the sequences ofHER3 (ERBB3; MIM 190151; 2065). These SMOs are used according to themethods of the invention to modulate splicing of HER3 pre-mRNA. In oneembodiment, a SMO of the invention functions to decrease HER3 expressionor function. In another embodiment, the invention includes apharmaceutical composition comprising a SMO of the invention, where thepharmaceutical composition of the invention comprises a SMO thatfunctions to decrease HER3 expression or function. In one aspect, theSMO contacts a HER3 pre-mRNA and modulates the splicing of the HER3pre-mRNA to favor expression of HER3Δ3, a variant in which exon 3 ofHER3 is deleted and is, thus, non-functional. In another aspect, the SMOcontacts a HER3 pre-mRNA and modulates the splicing of the HER3 pre-mRNAto favor expression of HER3Δ11, a variant in which exon 11 of HER3 isdeleted and the mature protein is non-functional. In still anotheraspect, the SMO contacts a HER pre-mRNA and modulates splicing of theHER3 pre-mRNA to favor inclusion of intron 3 of HER3, thus enhancingexpression of a truncated, non-functional protein.

Table 10 depicts exemplary SMOs for modulating splicing of HER3 pre-mRNAin order to either block a 3′ splice site of exon 3 or include intron 3,thereby increasing expression of a truncated protein in a cell.

TABLE 10 3′ to 5′ Splice modulating  SEQ oligonucleotides targeting IDexon 3 of HER3 NO. CGGUCGAGGCGAACUGAGUCGAGU 729 UGAGUCGAGUGGCcagucaaggg730 GGCGAACUGAGUCGAGUGGCca 731 GCGAACUGAGUCGAGUGGCcag 732CGAACUGAGUCGAGUGGCcagu 733 GAACUGAGUCGAGUGGCcaguc 734AACUGAGUCGAGUGGCcaguca 735 ACUGAGUCGAGUGGCcagucaa 736CUGAGUCGAGUGGCcagucaag 737 UGAGUCGAGUGGCcagucaagg 738GAGUCGAGUGGCcagucaaggg 739 GCGAACUGAGUCGAGUGGCca 740CGAACUGAGUCGAGUGGCcag 741 GAACUGAGUCGAGUGGCcagu 742AACUGAGUCGAGUGGCcaguc 743 ACUGAGUCGAGUGGCcaguca 744CUGAGUCGAGUGGCcagucaa 745 UGAGUCGAGUGGCcagucaag 746GAGUCGAGUGGCcagucaagg 747 AGUCGAGUGGCcagucaaggg 748 CGAACUGAGUCGAGUGGCca749 GAACUGAGUCGAGUGGCcag 750 AACUGAGUCGAGUGGCcagu 751ACUGAGUCGAGUGGCcaguc 752 CUGAGUCGAGUGGCcaguca 753 UGAGUCGAGUGGCcagucaa754 GAGUCGAGUGGCcagucaag 755 AGUCGAGUGGCcagucaagg 756GUCGAGUGGCcagucaaggg 757 GAACUGAGUCGAGUGGCca 758 AACUGAGUCGAGUGGCcag 759ACUGAGUCGAGUGGCcagu 760 CUGAGUCGAGUGGCcaguc 761 UGAGUCGAGUGGCcaguca 762GAGUCGAGUGGCcagucaa 763 AGUCGAGUGGCcagucaag 764 GUCGAGUGGCcagucaagg 765UCGAGUGGCcagucaaggg 766 AACUGAGUCGAGUGGCca 767 ACUGAGUCGAGUGGCcag 768CUGAGUCGAGUGGCcagu 769 UGAGUCGAGUGGCcaguc 770 GAGUCGAGUGGCcaguca 771AGUCGAGUGGCcagucaa 772 GUCGAGUGGCcagucaag 773 UCGAGUGGCcagucaagg 774CGAGUGGCcagucaaggg 775 ACUGAGUCGAGUGGCca 776 CUGAGUCGAGUGGCcag 777UGAGUCGAGUGGCcagu 778 GAGUCGAGUGGCcaguc 779 AGUCGAGUGGCcaguca 780GUCGAGUGGCcagucaa 781 UCGAGUGGCcagucaag 782 CGAGUGGCcagucaagg 783GAGUGGCcagucaaggg 784 CUGAGUCGAGUGGCca 785 UGAGUCGAGUGGCcag 786GAGUCGAGUGGCcagu 787 AGUCGAGUGGCcaguc 788 GUCGAGUGGCcaguca 789UCGAGUGGCcagucaa 790 CGAGUGGCcagucaag 791 GAGUGGCcagucaagg 792AGUGGCcagucaaggg 793 UGAGUCGAGUGGCca 794 GAGUCGAGUGGCcag 795AGUCGAGUGGCcagu 796 GUCGAGUGGCcaguc 797 UCGAGUGGCcaguca 798CGAGUGGCcagucaa 799 GAGUGGCcagucaag 800 AGUGGCcagucaagg 801GUGGCcagucaaggg 802

Table 11 depicts exemplary SMOs for modulating splicing of HER3 pre-mRNAin order to exclude exon 11 thereby increasing expression of anon-functional protein in a cell.

TABLE 11 3′ to 5′ Splice modulating oligonucleotides directed toexon 11 of HER3 [[Should refer SEQ to them ONLY as SMOs or oligo- IDnucleotides throughout.]] NO. cggagagagguuggggagucCAAU 803ggggagucCAAUGGACUUGUAGGU 804 gggagucCAAUGGACUUGUAGGUC 805cggagagagguuggggagucCAA 806 ggagucCAAUGGACUUGUAGGUC 807cggagagagguuggggagucCA 808 gagucCAAUGGACUUGUAGGUC 809agucCAAUGGACUUGUAGGUC 810 gucCAAUGGACUUGUAGGUC 811 ucCAAUGGACUUGUAGGUC812 ucCAAUGGACUUGUAGGU 813

F. Cyclophilin D

The present invention further provides SMOs based on the sequences ofCypD (PPID; MIM: 601753 GeneID: 5481). These SMOs are used according tothe methods of the invention to modulate splicing of CypD pre-mRNA. Inone embodiment, a SMO of the invention functions to decrease CypDexpression or function. In another embodiment, the invention includes apharmaceutical composition comprising a SMO of the invention, where thepharmaceutical composition of the invention comprises a SMO thatfunctions to decrease the CypD expression or function. In one aspect,the SMO contacts a CypD pre-mRNA and modulates the splicing of the CypDpre-mRNA to favor expression of CypDΔ1, a variant in which exon 1 ofCypD is deleted and is, thus, non-functional. In another aspect, the SMOcontacts an CypD pre-mRNA and modulates the splicing of the CypDpre-mRNA to favor expression of CypDΔ3, a variant in which exon 3 ofCypD is deleted and is, thus, non-functional.

Table 12 depicts exemplary SMOs for modulating splicing of CypD pre-mRNAin order to exclude exon 1 thereby decreasing expression of a functionalCypD protein in a cell.

TABLE 12 3′ to 5′ Splice modulating SEQ oligonucleotides directed  IDto targeting exon 1 of CypD NO. UGCAGACGUUCAGUUCUACAGCGU 814UGCAGACGUUCAGUUCUACAGCG 815 UGCAGACGUUCAGUUCUACAGC 816UGCAGACGUUCAGUUCUACAG 817 UGCAGACGUUCAGUUCUACA 818 UGCAGACGUUCAGUUCUAC819 AGACGUUCAGUUCUACAGCGUGGG 820 AGACGUUCAGUUCUACAGCGUGG 821AGACGUUCAGUUCUACAGCGUG 822 AGACGUUCAGUUCUACAGCGU 823AGACGUUCAGUUCUACAGCG 824 UGUAGCCUCCCCUCGCUCcacucg 825GUAGCCUCCCCUCGCUCcacucg 826 UAGCCUCCCCUCGCUCcacucg 827AGCCUCCCCUCGCUCcacucg 828 GCCUCCCCUCGCUCcacucg 829 CCUCCCCUCGCUCcacucg830 CUGUAGCCUCCCCUCGCUCcacuc 831 UGUAGCCUCCCCUCGCUCcacuc 832GUAGCCUCCCCUCGCUCcacuc 833 UAGCCUCCCCUCGCUCcacuc 834AGCCUCCCCUCGCUCcacuc 835 GCCUCCCCUCGCUCcacuc 836 CCUGUACCUCCCCUCGCUCcacu837 CUGUACCUCCCCUCGCUCcacu 838 UGUACCUCCCCUCGCUCcacu 839GUACCUCCCCUCGCUCcacu 840 UACCUCCCCUCGCUCcacu 841 ACCUCCCCUCGCUCcacu 842ACCUGUAGCCUCCCCUCGCUCcac 843 CCUGUAGCCUCCCCUCGCUCcac 844CUGUAGCCUCCCCUCGCUCcac 845 UGUAGCCUCCCCUCGCUCcac 846GUAGCCUCCCCUCGCUCcac 847 CACCUGUACCUCCCCUCGCUCca 848ACCUGUACCUCCCCUCGCUCca 849 CCUGUACCUCCCCUCGCUCca 850CUGUACCUCCCCUCGCUCca 851 UGUACCUCCCCUCGCUCca 852GCACCUGUAGCCUCCCCUCGCUCc 853 CACCUGUAGCCUCCCCUCGCUCc 854ACCUGUAGCCUCCCCUCGCUCc 855 CCUGUAGCCUCCCCUCGCUCc 856CUGUAGCCUCCCCUCGCUCc 857

Table 13 depicts exemplary SMOs for modulating splicing of CypD pre-mRNAin order to exclude exon 3 thereby decreasing expression of a functionalCypD protein in a cell.

TABLE 13 3′ to 5′ Splice modulating SEQ oligonucleotides directed IDto targeting exon 3 of CypD NO. acaucAAUAAUUCUUUAAAUACUA 858acaucAAUAAUUCUUUAAAUACU 859 acaucAAUAAUUCUUUAAAUAC 860acaucAAUAAUUCUUUAAAUA 861 acaucAAUAAUUCUUUAAAU 862 acaucAAUAAUUCUUUAAA863 caucAAUAAUUCUUUAAAUACUAA 864 caucAAUAAUUCUUUAAAUACUA 865caucAAUAAUUCUUUAAAUACU 866 caucAAUAAUUCUUUAAAUAC 867caucAAUAAUUCUUUAAAUA 868 caucAAUAAUUCUUUAAAU 869aucAAUAAUUCUUUAAAUACUAAG 870 aucAAUAAUUCUUUAAAUACUAA 871aucAAUAAUUCUUUAAAUACUA 872 aucAAUAAUUCUUUAAAUACU 873aucAAUAAUUCUUUAAAUAC 874 aucAAUAAUUCUUUAAAUA 875ucAAUAAUUCUUUAAAUACAAAGU 876 ucAAUAAUUCUUUAAAUACAAAG 877ucAAUAAUUCUUUAAAUACAAA 878 ucAAUAAUUCUUUAAAUACAA 879ucAAUAAUUCUUUAAAUACA 880 ucAAUAAUUCUUUAAAUAC 881cAAUAAUUCUUUAAAUACUAAGUC 882 cAAUAAUUCUUUAAAUACUAAGU 883cAAUAAUUCUUUAAAUACUAAG 884 cAAUAAUUCUUUAAAUACUAA 885cAAUAAUUCUUUAAAUACUA 886 cAAUAAUUCUUUAAAUAC 887 UUUAGUCUUACCCUGUCCACCUCU888 UUAGUCUUACCCUGUCCACCUCU 889 UAGUCUUACCCUGUCCACCUCU 890AGUCUUACCCUGUCCACCUCU 891 AGUCUUACCCUGUCCACCUC 892 AGUCUUACCCUGUCCACCU893 GUCUUACCCUGUCCACCUCUUUCA 894 UCUUACCCUGUCCACCUCUUUCA 895CUUACCCUGUCCACCUCUUUCA 896 UUACCCUGUCCACCUCUUUCA 897UACCCUGUCCACCUCUUUCA 898 ACCCUGUCCACCUCUUUCA 899 ACUUUUUAAACUUCUACUUU900 UUCUACUUUUAAAGGUAAUGUUCc 901 UCUACUUUUAAAGGUAAUGUUCc 902CUACUUUUAAAGGUAAUGUUCc 903 UACUUUUAAAGGUAAUGUUCc 904ACUUUUAAAGGUAAUGUUCc 905 CUUUUAAAGGUAAUGUUCc 906 CUACUUUUAAAGGUAAUGUUCca907 UACUUUUAAAGGUAAUGUUCca 908 ACUUUUAAAGGUAAUGUUCca 909CUUUUAAAGGUAAUGUUCca 910 UUUUAAAGGUAAUGUUCca 911 UUUAAAGGUAAUGUUCca 912CUACUUUUAAAGGUAAUGUUCcau 913 UACUUUUAAAGGUAAUGUUCcau 914ACUUUUAAAGGUAAUGUUCcau 915 CUUUUAAAGGUAAUGUUCcau 916UUUUAAAGGUAAUGUUCcau 917 UUUAAAGGUAAUGUUCcau 918

G. FOXM1

The present invention further provides SMOs based on the sequences ofFOXM1 (FOXM1; MIM: 602341; GeneID: 2305). These SMOs are used accordingto the methods of the invention to modulate splicing of FOXM1 pre-mRNA.In one embodiment, a SMO of the invention functions to decrease FOXM1expression. In another embodiment, the invention includes apharmaceutical composition comprising a SMO of the invention, where thepharmaceutical composition of the invention comprises a SMO thatfunctions to decrease the FOXM1 expression. In one aspect, the SMOcontacts a FOXM1 pre-mRNA and modulates the splicing of the FOXM1pre-mRNA to favor expression of FOXM1Δ3, a variant in which exon 3 ofFOXM1 D is excluded. In another aspect, the SMO contacts an FOXM1pre-mRNA and modulates the splicing of the FOXM1 pre-mRNA to favorexpression of FOXM1Δ6, a variant in which exon 6 of FOXM1 is excluded.

Table 14 depicts exemplary SMOs for modulating splicing of FOXM1pre-mRNA in order to exclude exon 3 thereby decreasing expression of afunctional FOXM1 protein in a cell.

TABLE 14 3′ to 5′ Splice modulating SEQ oligonucleotides directed IDto targeting Exon 3 of FOXM1 NO. GUAGGUCACCGAAGCUUUCUAC 919GUAGGUCACCGAAGCUUUCUA 920 GUAGGUCACCGAAGCUUUCU 921CCUCUUAACAGUGGACCUCGUC 922 CCUCUUAACAGUGGACCUCGU 923CUCUUAACAGUGGACCUCGU 924 CUCUUAACAGUGGACCUCG 925 CUCUUAACAGUGGACCUC 926ACCUCGUCGCUGUCCAAUUCca 927 CCUCGUCGCUGUCCAAUUCcau 928CUCGUCGCUGUCCAAUUCcacu 929 UCGUCGCUGUCCAAUUCcacuu 930CCUCGUCGCUGUCCAAUUCca 931 CUCGUCGCUGUCCAAUUCcac 932UCGUCGCUGUCCAAUUCcacu 933 CGUCGCUGUCCAAUUCcacuu 934GUCGCUGUCCAAUUCcacuua 935 UCGCUGUCCAAUUCcacuuaa 936 CUCGUCGCUGUCCAAUUCca937 UCGUCGCUGUCCAAUUCcac 938 CGUCGCUGUCCAAUUCcacu 939GUCGCUGUCCAAUUCcacuu 940 UCGCUGUCCAAUUCcacuua 941 CGCUGUCCAAUUCcacuuaa942 UCGUCGCUGUCCAAUUCca 943 CGUCGCUGUCCAAUUCcac 944 GUCGCUGUCCAAUUCcacu945 UCGCUGUCCAAUUCcacuu 946 CGCUGUCCAAUUCcacuua 947 GCUGUCCAAUUCcacuuaa948 CGUCGCUGUCCAAUUCca 949 GUCGCUGUCCAAUUCcac 950 UCGCUGUCCAAUUCcacu 951CGCUGUCCAAUUCcacuu 952 GCUGUCCAAUUCcacuua 953

Table 15 depicts exemplary SMOs for modulating splicing of FOXM1pre-mRNA in order to exclude exon 6 decreasing expression of afunctional FOXM1 protein in a cell.

TABLE 15 3′ to 5′ Splice modulating SEQ oligonucleotides directed IDto targeting Exon 6 of FOXM1 NO. GGCGGUGGUCGGCGGUGGUCGG  954GGCGGUGGUCGGCGGUGGUCGGU  955 GGCGGUGGUCGGCGGUGGUCGG  956GGCGGUGGUCGGCGGUGGUCG  957 GGCGGUGGUCGGCGGUGGUC  958 GGCGGUGGUCGGCGGUGGU 959 cGGCGGUGGUCGGUGACCUGGGUC  960 cGGCGGUGGUCGGUGACCUGGGU  961cGGCGGUGGUCGGUGACCUGGG  962 cGGCGGUGGUCGGUGACCUGG  963cGGCGGUGGUCGGUGACCUG  964 cGGCGGUGGUCGGUGACCU  965ccGGCGGUGGUCGGCGGUGGUCGG  966 ccGGCGGUGGUCGGCGGUGGUCG  967ccGGCGGUGGUCGGCGGUGGUC  968 ccGGCGGUGGUCGGCGGUGGU  969ccGGCGGUGGUCGGCGGUGG  970 ccGGCGGUGGUCGGCGGUG  971accGGCGGUGGUCGGCGGUGGUCG  972 accGGCGGUGGUCGGCGGUGGUC  973accGGCGGUGGUCGGCGGUGGU  974 accGGCGGUGGUCGGCGGUGG  975accGGCGGUGGUCGGCGGUG  976 accGGCGGUGGUCGGCGGU  977gaccGGCGGUGGUCGGCGGUGGUC  978 gaccGGCGGUGGUCGGCGGUGGU  979gaccGGCGGUGGUCGGCGGUGG  980 gaccGGCGGUGGUCGGCGGUG  981gaccGGCGGUGGUCGGCGGU  982 gaccGGCGGUGGUCGGCGG  983ggaccGGCGGUGGUCGGCGGUGGU  984 ggaccGGCGGUGGUCGGCGGUGG  985ggaccGGCGGUGGUCGGCGGUG  986 ggaccGGCGGUGGUCGGCGGU  987ggaccGGCGGUGGUCGGCGG  988 ggaccGGCGGUGGUCGGCG  989cggaccGGCGGUGGUCGGCGGUGG  990 cggaccGGCGGUGGUCGGCGGUG  991cggaccGGCGGUGGUCGGCGGU  992 cggaccGGCGGUGGUCGGCGG  993cggaccGGCGGUGGUCGGCG  994 cggaccGGCGGUGGUCGGC  995CGGUGGUCGGUGACCUGGGUCCCA  996 CGGUGGUCGGUGACCUGGGUCCC  997CGGUGGUCGGUGACCUGGGUCC  998 CGGUGGUCGGUGACCUGGGUC  999CGGUGGUCGGUGACCUGGGU 1000 GGUGGUCGGUGACCUGGGUCCCAG 1001GGUGGUCGGUGACCUGGGUCCCA 1002 GGUGGUCGGUGACCUGGGUCCC 1003GGUGGUCGGUGACCUGGGUCC 1004 GGUGGUCGGUGACCUGGGUC 1005GUGGUCGGUGACCUGGGUCCCAGA 1006 GUGGUCGGUGACCUGGGUCCCAG 1007GUGGUCGGUGACCUGGGUCCCA 1008 GUGGUCGGUGACCUGGGUCCC 1009GUGGUCGGUGACCUGGGUCC 1010 UGGGUCCCAGAGGUGUUAACGGGC 1011UGGGUCCCAGAGGUGUUAACGGG  1012 UGGGUCCCAGAGGUGUUAACGG 1013UGGGUCCCAGAGGUGUUAACG 1014 UGGGUCCCAGAGGUGUUAAC 1015GGGUCCCAGAGGUGUUAACGGGCU 1016 GGGUCCCAGAGGUGUUAACGGGC 1017GGGUCCCAGAGGUGUUAACGGG 1018 GGGUCCCAGAGGUGUUAACGG 1019GGGUCCCAGAGGUGUUAACG 1020 CCCAGAGGUGUUAACGGGCUCGUG 1021CCCAGAGGUGUUAACGGGCUCGU 1022 CCCAGAGGUGUUAACGGGCUCG 1023CCCAGAGGUGUUAACGGGCUC 1024 CCCAGAGGUGUUAACGGGCU 1025AGAGGUGUUAACGGGCUCGUGAAC 1026 AGAGGUGUUAACGGGCUCGUGAA 1027AGAGGUGUUAACGGGCUCGUGA 1028 AGAGGUGUUAACGGGCUCGUG 1029AGAGGUGUUAACGGGCUCGU 1030 GAGGUGUUAACGGGCUCGUGAACC 1031GAGGUGUUAACGGGCUCGUGAAC 1032 GAGGUGUUAACGGGCUCGUGAA 1033GAGGUGUUAACGGGCUCGUGA 1034 GAGGUGUUAACGGGCUCGUG 1035GUUAACGGGCUCGUGAACCUUAGU 1036 GUUAACGGGCUCGUGAACCUUAG 1037GUUAACGGGCUCGUGAACCUUA 1038 GUUAACGGGCUCGUGAACCUU 1039GUUAACGGGCUCGUGAACCU 1040 GUUAACGGGCUCGUGAACC 1041UUAACGGGCUCGUGAACCUUAGUc 1042 UAACGGGCUCGUGAACCUUAGUc 1043AACGGGCUCGUGAACCUUAGUc 1044 ACGGGCUCGUGAACCUUAGUc 1045CGGGCUCGUGAACCUUAGUc 1046 GGGCUCGUGAACCUUAGUc 1047UAACGGGCUCGUGAACCUUAGUca 1048 AACGGGCUCGUGAACCUUAGUca 1049ACGGGCUCGUGAACCUUAGUca 1050 CGGGCUCGUGAACCUUAGUca 1051GGGCUCGUGAACCWAGUca 1052 GGCUCGUGAACCUUAGUca 1053AACGGGCUCGUGAACCUUAGUcau 1054 ACGGGCUCGUGAACCUUAGUcau 1055CGGGCUCGUGAACCUUAGUcau 1056 GGGCUCGUGAACCUUAGUcau 1057GGCUCGUGAACCUUAGUcau 1058 GCUCGUGAACCUUAGUcau 1059ACGGGCUCGUGAACCUUAGUcauu 1060 CGGGCUCGUGAACCUUAGUcauu 1061GGGCUCGUGAACCUUAGUcauu 1062 GGCUCGUGAACCUUAGUcauu 1063GCUCGUGAACCUUAGUcauu 1064 CUCGUGAACCUUAGUcauu 1065CGGGCUCGUGAACCUUAGUcauuc 1066 GGGCUCGUGAACC'UUAGUcauuc 1067GGCUCGUGAACCUUAGUcauuc 1068 GCUCGUGAACCUUAGUcauuc 1069CUCGUGAACCUUAGUcauuc 1070 UCGUGAACCUUAGUcauuc 1071GGGCUCGUGAACCUUAGUcauucc 1072 GGCUCGUGAACCUUAGUcauucc 1073GCUCGUGAACCUUAGUcauucc 1074 CUCGUGAACCUUAGUcauucc 1075UCGUGAACCUUAGUcauucc 1076 CGUGAACCUUAGUcauucc 1077GGCUCGUGAACCUUAGUcauucca 1078 GCUCGUGAACCUUAGUcauucca 1079CUCGUGAACCUUAGUcauucca 1080 UCGUGAACCUUAGUcauucca 1081CGUGAACCUUAGUcauucca 1082 GUGAACCUUAGUcauucca 1083GCUCGUGAACCUUAGUcauuccaa 1084 CUCGUGAACCUUAGUcauuccaa 1085UCGUGAACCUUAGUcauuccaa 1086 CGUGAACCUUAGUcauuccaa 1087GUGAACCUUAGUcauuccaa 1088 UGAACCUUAGUcauuccaa 1089

It will be appreciated by the skilled artisan that a SMO useful inpracticing the methods of the invention should not be considered to belimited to those SMO sequences explicitly recited herein, but rathershould be considered to include any SMO sufficiently complementary to atarget pre-mRNA in such a way as to modulate its splicing. The inventionalso encompasses all derivatives, variants, and modifications of theSMOs of the invention, as described elsewhere herein.

Oligonucleotides of the invention are of any size and/or chemicalcomposition sufficient to specifically bind to a target RNA (e.g.,pre-mRNA). In exemplary embodiments, the oligonucleotides of theinvention are oligonucleotides of between about 5-300 nucleotides (ormodified nucleotides), preferably between about 10-100 nucleotides (ormodified nucleotides; e.g., ribonucleotides or modifiedribonucleotides), for example, between about 15-35, e.g., about 15-20,20-25, 25-30, 30-35 (31, 32, 33, 34, 35), or 35-40 nucleotides (ormodified nucleotides; e.g., ribonucleotides or modifiedribonucleotides).

Synthesis of SMOs

An oligonucleotide of the invention, i.e. the SMO, can be synthesizedusing any procedure known in the art, including chemical synthesis,enzymatic ligation, organic synthesis, and biological synthesis.

In one embodiment, an RNA molecule, e.g., SMO, is prepared chemically.Methods of synthesizing RNA and DNA molecules are known in the art, inparticular, the chemical synthesis methods as described in Verma andEckstein (1998) Annul Rev. Biochem. 67:99-134. RNA can be purified froma mixture by extraction with a solvent or resin, precipitation,electrophoresis, chromatography, or a combination thereof.Alternatively, the RNA may be used with no or a minimum of purificationto avoid losses due to sample processing.

Modifications of SMOs

In a preferred aspect, the oligonucleotides of the present invention(i.e. SMOs) are modified to improve stability in serum or growth mediumfor cell cultures, or otherwise to enhance stability during delivery tosubjects and/or cell cultures. In order to enhance the stability, the3′-residues may be stabilized against degradation, e.g., they may beselected such that they consist of purine nucleotides, particularlyadenosine or guanosine nucleotides. Alternatively, substitution ofpyrimidine nucleotides by modified analogues, e.g., substitution ofuridine by 2′-deoxythymidine can be tolerated without affecting theefficiency of oligonucleotide reagent-induced modulation of splice siteselection. For example, the absence of a 2′ hydroxyl may significantlyenhance the nuclease resistance of the oligonucleotides in tissueculture medium.

In an embodiment of the present invention the oligonucleotides, e.g.,SMOs, may contain at least one modified nucleotide analogue. Thenucleotide analogues may be located at positions where thetarget-specific activity, e.g., the splice site selection modulatingactivity is not substantially effected, e.g., in a region at the 5′-endand/or the 3′-end of the oligonucleotide molecule. Particularly, theends may be stabilized by incorporating modified nucleotide analogues.

Preferred nucleotide analogues include sugar- and/or backbone-modifiedribonucleotides (i.e., include modifications to the phosphate-sugarbackbone). For example, the phosphodiester linkages of natural RNA maybe modified to include at least one of a nitrogen or sulfur heteroatom.In preferred backbone-modified ribonucleotides the phosphoester groupconnecting to adjacent ribonucleotides is replaced by a modified group,e.g., of phosphothioate group. In preferred sugar-modifiedribonucleotides, the 2′ OH-group is replaced by a group selected fromCH₃, H, OR, R, halo, SH, SR, NH₂, NHR, NR₂ or ON, wherein R is C₁-C₆alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I. In a preferredembodiment, the 2′ OH-group is replaced by CH₃.

Also preferred are nucleobase-modified ribonucleotides, i.e.,ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases may bemodified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to phosphorothioatederivatives and acridine substituted nucleotides, 2′O-methylsubstitutions, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine, uridine and/or cytidine modified at the5-position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosineand/or guanosines modified at the 8 position, e.g., 8-bromo guanosine;deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-alkylatednucleotides, e.g., N6-methyl adenosine. It should be noted that theabove modifications may be combined. Oligonucleotides of the inventionalso may be modified with chemical moieties (e.g., cholesterol) thatimprove the in vivo pharmacological properties of the oligonucleotides.

Within the oligonucleotides (e.g., oligoribonucleotides) of theinvention, as few as one and as many as all nucleotides of theoligonucleotide can be modified. For example, a 20-mer oligonucleotide(e.g., oligoribonucleotide) of the invention may contain 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 modifiednucleotides. In preferred embodiments, the modified oligonucleotides(e.g., oligoribonucleotides) of the invention will contain as fewmodified nucleotides as are necessary to achieve a desired level of invivo stability and/or bioaccessibility while maintaining costeffectiveness. An SMOs of the invention include oligonucleotidessynthesized to include any combination of modified bases disclosedherein in order to optimize function. In one embodiment, a SMO of theinvention comprises at least two different modified bases. In anotherembodiment, a SMO of the invention may comprise alternating 2′O-methylsubstitutions and LNA bases.

An oligonucleotide of the invention can be an α-anomeric nucleic acidmolecule. An α-anomeric nucleic acid molecule forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual α-units, the strands run parallel to each other (Gaultier et al.,1987, Nucleic Acids Res. 15:6625-6641). The oligonucleotide can alsocomprise a 2′-o-methylribonucleotide (Inoue et al., 1987, Nucleic AcidsRes. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., 1987,FEBS Lett. 215:327-330).

In various embodiments, the oligonucleotides of the invention can bemodified at the base moiety, sugar moiety or phosphate backbone toimprove, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacid molecules can be modified to generate peptide nucleic acidmolecules (see Hyrup et al., 1996, Bioorganic & Medicinal Chemistry4(1): 5-23). As used herein, the terms “peptide nucleic acids” or “PNAs”refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribosephosphate backbone is replaced by a pseudopeptide backbone and only thefour natural nucleobases are retained. The neutral backbone of PNAs hasbeen shown to allow for specific hybridization to DNA and RNA underconditions of low ionic strength. The synthesis of PNA oligomers can beperformed using standard solid phase peptide synthesis protocols asdescribed in Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996)Proc. Natl. Acad. Sci. USA 93:14670-675.

In another embodiment, PNAs can be modified, e.g., to enhance theirstability or cellular uptake, by attaching lipophilic or other helpergroups to PNA, by the formation of PNA-DNA chimeras, or by the use ofliposomes or other techniques of drug delivery known in the art. Forexample, PNA-DNA chimeras can be generated which can combine theadvantageous properties of PNA and DNA. Such chimeras allow DNArecognition enzymes, e.g., RNase H and DNA polymerases, to interact withthe DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup, 1996, supra). Thesynthesis of PNA-DNA chimeras can be performed as described in Hyrup(1996), supra, and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63.For example, a DNA chain can be synthesized on a solid support usingstandard phosphoramiditea coupling chemistry and modified nucleosideanalogs. Compounds such as 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite can be used as a link between the PNA and the 5′ end ofDNA (Mag et al., 1989, Nucleic Acids Res. 17:5973-88). PNA monomers arethen coupled in a step-wise manner to produce a chimeric molecule with a5′ PNA segment and a 3′ DNA segment (Finn et al., 1996, Nucleic AcidsRes. 24(17): 3357-63). Alternatively, chimeric molecules can besynthesized with a 5′ DNA segment and a 3′ PNA segment (Peterser et al.,1975, Bioorganic Med. Chem. Lett. 5: 1119-11124).

The oligonucleotides of the invention can also be formulated asmorpholino oligonucleotides. In such embodiments, the riboside moiety ofeach subunit of an oligonucleotide of the oligonucleotide is convertedto a morpholine moiety (morpholine=C₄H₉NO; refer to Heasman, J. 2002Developmental Biology 243, 209-214, the entire contents of which areincorporated herein by reference).

A further preferred oligonucleotide modification includes Locked NucleicAcids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′carbon atom of the sugar ring thereby forming a bicyclic sugar moiety.The linkage is preferably a methelyne (—CH₂—)_(n) group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226, theentire contents of which are incorporated by reference herein.

In other embodiments, the oligonucleotide can include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al., 1987, Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO 89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (see, e.g., Krolet al., 1988, Bio/Techniques 6:958-976) or intercalating agents (see,e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide can be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The invention also includes molecular beacon nucleic acid moleculeshaving at least one region which is complementary to a nucleic acidmolecule of the invention, such that the molecular beacon is useful forquantitating the presence of the nucleic acid molecule of the inventionin a sample. A “molecular beacon” nucleic acid is a nucleic acidmolecule comprising a pair of complementary regions and having afluorophore and a fluorescent quencher associated therewith. Thefluorophore and quencher are associated with different portions of thenucleic acid in such an orientation that when the complementary regionsare annealed with one another, fluorescence of the fluorophore isquenched by the quencher. When the complementary regions of the nucleicacid molecules are not annealed with one another, fluorescence of thefluorophore is quenched to a lesser degree. Molecular beacon nucleicacid molecules are described, for example, in U.S. Pat. No. 5,876,930.

The target RNA (e.g., pre-mRNA) splice-modifying reaction guided byoligonucleotides of the invention is highly sequence specific. Ingeneral, oligonucleotides containing nucleotide sequences perfectlycomplementary to a portion of the target RNA are preferred for blockingof the target RNA. However, 100% sequence complementarity between theoligonucleotide and the target RNA is not required to practice thepresent invention. Thus, the invention may tolerate sequence variationsthat might be expected due to genetic mutation, strain polymorphism, orevolutionary divergence. For example, oligonucleotide sequences withinsertions, deletions, and single point mutations relative to the targetsequence may also be effective for inhibition. Alternatively,oligonucleotide sequences with nucleotide analog substitutions orinsertions can be effective for blocking.

Greater than 70% sequence identity (or complementarity), e.g., 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% oreven 100% sequence identity, and any and all whole or partial incrementsthere between the oligonucleotide and the target RNA, e.g., targetpre-mRNA, is preferred.

Sequence identity, including determination of sequence complementarityfor nucleic acid sequences, may be determined by sequence comparison andalignment algorithms known in the art. To determine the percent identityof two nucleic acid sequences (or of two amino acid sequences), thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in the first sequence or second sequence for optimalalignment). The nucleotides (or amino acid residues) at correspondingnucleotide (or amino acid) positions are then compared. When a positionin the first sequence is occupied by the same residue as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=number of identical positions/totalnumber of positions×100), optionally penalizing the score for the numberof gaps introduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the alignment generated over a certainportion of the sequence aligned having sufficient identity but not overportions having low degree of identity (i.e., a local alignment). Apreferred, non-limiting example of a local alignment algorithm utilizedfor the comparison of sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithmis incorporated into the BLAST programs (version 2.0) of Altschul, etal. (1990) J. Mol. Biol. 215:403-10.

In another embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the length ofthe aligned sequences (i.e., a gapped alignment). To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. In another embodiment, the alignment is optimized byintroducing appropriate gaps and percent identity is determined over theentire length of the sequences aligned (i.e., a global alignment). Apreferred, non-limiting example of a mathematical algorithm utilized forthe global comparison of sequences is the algorithm of Myers and Miller,CABIOS (1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM 120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. Alternatively, the oligonucleotidemay be defined functionally as a nucleotide sequence (or oligonucleotidesequence) a portion of which is capable of hybridizing with the targetRNA (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C.hybridization for 12-16 hours; followed by washing). Additionalpreferred hybridization conditions include hybridization at 70° C. in1×SSC or 50° C. in 1×SSC, 50% formamide followed by washing at 70° C. in0.3×SSC or hybridization at 70° C. in 4×SSC or 50° C. in 4×SSC, 50%formamide followed by washing at 67° C. in 1×SSC. The hybridizationtemperature for hybrids anticipated to be less than 50 base pairs inlength should be 5-10° C. less than the melting temperature (Tm) of thehybrid, where Tm is determined according to the following equations. Forhybrids less than 18 base pairs in length, Tm(° C.).=2(number of A+Tbases)+4(number of G+C bases). For hybrids between 18 and 49 base pairsin length, Tm(° C.).=81.5+16.6(log 10[Na⁺])+0.41(% G+C)−(600/N), where Nis the number of bases in the hybrid, and [Na⁺] is the concentration ofsodium ions in the hybridization buffer ([Na⁺] for 1×SSC=0.165 M).Additional examples of stringency conditions for polynucleotidehybridization are provided in Sambrook, et al., 2001, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., chapters 9 and 11, and Current Protocols in MolecularBiology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons, Inc.,sections 2.10 and 6.3-6.4, incorporated herein by reference. The lengthof the identical nucleotide sequences may be at least about 10, 12, 15,17, 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, 45, 47 or 50 bases.

II. Methods

The present invention provides compositions and methods for modulatingpre-mRNA splicing using a SMO of the invention to abrogatedisease-causing mutations in a protein. An SMO of the invention maymodulate pre-mRNA splicing by blocking cryptic splice sites, removing anexon, including an exon, or shifting the reading frame of the pre-mRNAin order to alter protein isoform expression.

Accordingly, the present invention provides compositions and methods oftreating a subject at risk of, susceptible to, or having a disease,disorder, or condition associated with aberrant or unwanted targetpre-mRNA expression or function. In one embodiment, a target pre-mRNA ofthe invention is any aberrantly spliced or unwanted pre-mRNA encoding aprotein that results in, causes, produces, or pre-disposes a subject toa disease or disorder. In another embodiment, aberrant splicing of atarget pre-mRNA if the invention is not a cause of a disease ordisorder, but modulation of the target pre-mRNA reduces at least onesymptom of the disease or disorder.

In another embodiment, the invention provides a method of preventing ina subject, a disease, disorder, or condition associated with aberrant orunwanted pre-mRNA splicing of a protein and altered protein expressionor function, the method comprising administering to the subject apharmaceutical composition comprising a SMO, or vector, or transgeneencoding same.

A target pre-mRNA of the invention is any pre-mRNA that is abnormallyspliced or a pre-mRNA whose altered activity is likely to have abeneficial effect on a subject. In one embodiment, a target pre-mRNA ofthe invention comprises a 5-HT2C receptor. In yet another embodiment, atarget pre-mRNA of the invention is an aberrantly spliced 5-HT2CRpre-mRNA in a subject that results in a truncated, non-functional 5-HT2Creceptor.

In yet another embodiment, a target pre-mRNA of the invention is an AMPAglutamate receptor (GluR) subunit comprising GluR1, GluR2, Glur3, GluR4,or any combination thereof. In a further embodiment, a target pre-mRNAof the invention is an AMPA glutamate receptor (GluR) subunit comprisingGluR1, GluR2, Glur3, GluR4, or any combination thereof where it isdesirable to alter the ratio of flip and flop isoforms of any one of, orany combination of these GluRs. In yet another embodiment, a targetpre-mRNA of the invention is an aberrantly spliced GluR pre-mRNA in asubject that results in a truncated, non-functional glutamate receptor.

In still another embodiment, a target pre-mRNA of the invention is OGA.

In yet another embodiment of the invention, a target pre-mRNA of theinvention is Aph1B. In another embodiment, a target pre-mRNA of theinvention is HER3. In still another embodiment, a target pre-mRNA of theinvention is FOXM1. In yet another embodiment, a target pre-mRNA of theinvention is CypD.

Subjects at risk for a disease which is caused or contributed to byaberrant or unwanted target pre-mRNA expression or activity can beidentified by, for example, any or a combination of diagnostic orprognostic assays known in the art. Administration of a prophylacticagent comprising a SMO can occur prior to the manifestation of symptomscharacteristic of the target pre-mRNA aberrancy, such that a disease ordisorder is prevented or, alternatively, delayed in its progression.

The invention encompasses methods of modulating target pre-mRNA splicingand thus expression or activity of the specified protein for therapeuticpurposes. In an exemplary embodiment, the modulatory method of theinvention involves contacting a cell capable of expressing a targetpre-mRNA with a pharmaceutical composition comprising a SMO or vector ortransgene encoding same, that is specific for the target pre-mRNA (e.g.,is specific for the pre-mRNA) such that expression or one or more of theactivities of target pre-mRNA is modulated. These modulatory methods canbe performed in vitro (e.g., by culturing the cell with the agent) or,alternatively, in vivo (e.g., by administering the agent to a subject).As such, the present invention provides methods of treating a subjectafflicted with a disease or disorder characterized by aberrant splicingof a target pre-mRNA molecule resulting in deleterious proteinexpression or activity.

A. Method of Modulating 5-HT2C Receptor Pre-mRNA Splicing

In one embodiment, the present invention provides a method of modulating5-HT2C receptor pre-mRNA splicing using a SMO to mimic the function ofthe snoRNA, HBII-52, in a subject. The method comprises administering aSMO of the invention, or a composition comprising a SMO of theinvention, to a subject, wherein the SMO contacts 5-HT2CR pre-mRNA andmodulates the splicing of the 5-HT2CR pre-mRNA to include exon 5b in themature mRNA.

Diseases and disorders where increasing 5-HT2CR expression is believedto provide a therapeutic benefit to the subject afflicted with thedisease include, but are not limited to, PWS and Angelman Syndrome(Kishore et al., 2006, Cold Spring Harbor Symp. Quant. Biol. 71:329-334; Kishore et al., 2006, Science, 311: 230-232; Sridhar et al.,2008, J. Biomed. Sci., 15: 697-705); hyperphagia induced obesity (Dunlopet al., 2006, CNS Drug Rev., 12: 167-177; Nilsson, 2006, J. Med. Chem.,49: 4023-4034); obsessive/compulsive disorder (Flaisher-Grinberg et al.,2008, Int. J. Neuropsychopharmacology, 11: 811-825); depression,including psychotic depression, major depressive disorder, bipolardisorder (Rosenzweig-Lipson et al., 2007, Psychopharmacology (Berl),192: 159-170; Dunlop et al., 2006, CNS Drug Rev., 12: 167-177;Rosenzweig-Lipson et al., 2007, Drug News Perspect., 20: 565-571); sleepimpairment (Monti et al., 2008, Prog. Brain Res., 172: 625-646); autism(Tandon et al., 2008, Mol. Med., 105: 79-84); epilepsy (Bagdy et al.,2007, J. Neurochem., 100: 857-873; Tupal et al., 2006, Epilepsia, 47:21-26); schizophrenia (Rosenzweig-Lipson et al., 2007, Drug NewsPerspect., 20: 565-571); Parkinson's disease (Di et al., 2006, Curr.Med. Chem., 13: 3069-3081); drug addiction (Bubar et al., 2008, Prog.Brain Res., 172: 319-346); spinal cord injury or traumatic brain injury(Kao et al., 2006, Brain Res., 1112: 159-168); neuopathic pain (Nakae etal., 2008, Eur. J. Neurosci. 27: 2373-2379; Nakae et al., 2008,Neurosci. Res., 60: 228-231); diabetes (Wade et al., 2008,Endocrinology, 149: 955-961); Alzheimer's disease (Pritchard et al.,2008, Neurobiol. Aging, 29: 341-347; Arjona et al., 2002, Brain Res.,951: 135-140), and chronic pain.

In another embodiment, the present invention provides a method oftreating a subject afflicted with PWS. The method comprisesadministering a SMO of the invention, or a composition comprising a SMOof the invention, to a subject afflicted with PWS, wherein the SMOcontacts 5-HT2CR pre-mRNA and modulates the splicing of the 5-HT2CRpre-mRNA to include exon 5b from the mRNA, thereby resulting inexpression of a full-length, functional 5-HT2CR protein in the subject.

In yet another embodiment, the present invention provides a method oftreating a subject afflicted with a 5-HT2CR splicing defect, where thedefect results in a non-functional truncated 5-HT2C receptor thatincludes exon 5a, but not exon 5b. The method comprises administering aSMO of the invention, or a composition comprising a SMO of the inventionto a subject afflicted with a 5-HT2CR splicing defect, wherein the SMOcontacts 5-HT2CR pre-mRNA and modulates the splicing of the 5-HT2CRpre-mRNA, thereby resulting in expression of a full-length, functional5-HT2CR protein in the subject.

In still another embodiment, the present invention provides a method oftreating a subject afflicted with hyperphagia. In one aspect, thehyperphagia is caused by a 5-HT2CR splicing defect. In another aspect,the hyperphagia is not caused by a 5-HT2CR splicing defect, but thesubject afflicted with hyperphagia experiences a therapeutic benefitfrom increasing expression of the 5-HT2CR. The method comprisesadministering a SMO of the invention, or a composition comprising a SMOof the invention, to a subject afflicted with hyperphagia, wherein theSMO contacts 5-HT2CR pre-mRNA and modulates the splicing of the 5-HT2CRpre-mRNA, thereby resulting in increased expression of a full-length,functional 5-HT2CR protein and reducing hyperphagia in the subject.

In yet another embodiment, the present invention provides a method oftreating a subject afflicted with obsessive-compulsive disorder (OCD),or a subject afflicted with the symptoms of OCD, In one aspect, the OCDis caused by a 5-HT2CR splicing defect. In another aspect, the OCD isnot caused by a 5-HT2CR splicing defect, but the subject afflicted withOCD experiences a therapeutic benefit from increasing expression of the5-HT2CR. The method comprises administering a SMO of the invention, or acomposition comprising a SMO of the invention, to a subject afflictedwith OCD, wherein the SMO contacts 5-HT2CR pre-mRNA and modulates thesplicing of the 5-HT2CR pre-mRNA, thereby resulting in expression of afull-length, functional 5-HT2CR protein and a reductions of the symptomsof OCD in the subject.

B. Method of Modulating GluR Receptor Pre-mRNA Splicing

In one embodiment, the present invention provides a method of treating asubject afflicted with a GluR splicing defect, where the defect resultsin a non-functional GluR. The method comprises administering a SMO ofthe invention, or a composition comprising a SMO of the invention to asubject afflicted with a GluR splicing defect, wherein the SMO contactsGluR pre-mRNA and modulates the splicing of the GluR pre-mRNA, therebyresulting in expression of a full-length, functional GluR protein in thesubject. A skilled artisan will appreciate that the method may be usedto modulate splicing of a GluR1, GluR2, GluR3, or Glur4 subunit, as wellas any combination thereof.

In another embodiment, the present invention provides a method ofmodulating splicing of a GluR receptor pre-mRNA using a SMO to decreasethe GluR flip isoform expression in a subject. The method comprisesadministering a SMO of the invention, or a composition comprising a SMOof the invention, to a subject, wherein the SMO contacts a GluR pre-mRNAand modulates the splicing of the GluR to decrease the GluR flip isoformexpression and in the subject. A skilled artisan will appreciate thatthe method may be used to modulate splicing of a GluR1, GluR2, GluR3, orGlur4 subunit, as well as any combination thereof.

In yet another embodiment, the present invention provides a method oftreating a subject afflicted with a GluR splicing defect, where thedeficit results in a decreased flip:flop isoform ratio for a GluRsubunit. The method comprises administering a SMO of the invention, or acomposition comprising a SMO of the invention, to a subject afflictedwith an abnormal flip:flop ratio, wherein the SMO contacts GluR pre-mRNAand modulates the splicing of the GluR pre-mRNA, thereby resulting indecreased flip:flop isoform ratio for a GluR subunit. A skilled artisanwill appreciate that the method may be used to modulate splicing of aGluR1, GluR2, GluR3, or Glur4 subunit, as well as any combinationthereof.

In still another embodiment, the present invention provides a method oftreating a subject afflicted with amyotrophic lateral sclerosis (ALS;Sandyk, R., 2006, Int. J. Neurosci. 116: 775-826; Ionov, I. D., 2007,Amyotroph. Lateral Scler. 8:260-265). The method comprises administeringa SMO of the invention, or a composition comprising a SMO of theinvention, to a subject, wherein the SMO contacts a GluR pre-mRNA andmodulates the splicing of the GluR pre-mRNA to decrease the GluR flipisoform expression and/or decrease the GluR flip/flop isoform ratio inthe subject.

In another embodiment, the present invention provides a method oftreating a subject afflicted with epilepsy. The method comprisesadministering a SMO of the invention, or a composition comprising a SMOof the invention, to a subject, wherein the SMO contacts a GluR pre-mRNAand modulates the splicing of the GluR pre-mRNA to decrease the GluRflip isoform expression and/or decrease the GluR flip/flop isoformratio.

In yet another embodiment, the present invention provides a method ofdecreasing neuronal excitability in a subject. The method comprisesadministering a SMO of the invention, or a composition comprising a SMOof the invention to a subject afflicted with neuronal excitotoxity,wherein the SMO contacts GluR pre-mRNA and modulates the splicing of theGluR pre-mRNA, thereby resulting in decreased flip:flop isoform ratiofor a GluR subunit. A skilled artisan will appreciate that the methodmay be used to modulate splicing of a GluR1, GluR2, GluR3, or Glur4subunit, as well as any combination thereof.

In yet another embodiment, the present invention provides a method ofdecreasing a Ca^(2±)-conductance through a GluR in a subject. The methodcomprises administering a SMO of the invention, or a compositioncomprising a SMO of the invention to a subject, wherein the SMO contactsGluR pre-mRNA and modulates the splicing of the GluR pre-mRNA, therebyresulting in a decreased Ca²⁺-conductance through an AMPA channel in asubject. A skilled artisan will appreciate that the method may be usedto modulate splicing of a GluR1, GluR2, GluR3, or Glur4 subunit, as wellas any combination thereof.

In yet another embodiment, the present invention provides a method ofincreasing GluR desensitization in a subject. The method comprisesadministering a SMO of the invention, or a composition comprising a SMOof the invention to a subject, wherein the SMO contacts GluR pre-mRNAand modulates the splicing of the GluR pre-mRNA to decrease the GluRflip isoform expression and/or decrease the GluR flip/flop isoformratio, thereby resulting in a increased AMPA channel desensitization ina subject. A skilled artisan will appreciate that the method may be usedto modulate splicing of a GluR1, GluR2, GluR3, or Glur4 subunit, as wellas any combination thereof.

C. Method of Modulating OGA Receptor Pre-mRNA Splicing

In another embodiment, the present invention provides a method ofmodulating splicing of OGA pre-mRNA using a SMO to decrease expressionor functionality of OGA in a subject. The method comprises administeringa SMO of the invention, or a composition comprising a SMO of theinvention, to a subject, wherein the SMO contacts an OGA pre-mRNA andmodulates the splicing of the OGA pre-mRNA to favor expression ofnaturally occurring splice variants which have reduced catalyticactivity. In one aspect, an alternative splice variant of OGA withreduced catalytic activity comprises OGA10t, a read through variantwhich results in 15 amino acids being added from intron 10. In anotheraspect, the method comprises administering a SMO of the invention, or apharmaceutical composition comprising a SMO of the invention, to asubject, wherein the SMO contacts an OGA pre-mRNA and modulates thesplicing of the OGA pre-mRNA to favor expression of a non-naturalalternative splice variant of OGA with reduced catalytic activity. Inone aspect, an alternative splice variant of OGA with reduced catalyticactivity comprises OGAΔ10 wherein exon 10 of the gene is excluded. Inanother aspect, an alternative splice variant of OGA with reducedcatalytic activity comprises OGAΔ8 wherein exon 8 of the OGA gene isexcluded. Diseases and disorders where decreasing OGA expression isbelieved to provide a therapeutic benefit to the subject afflicted withthe disease include, but are not limited to, Alzheimer's Disease.

In another embodiment, the present invention provides a method oftreating a subject afflicted with Alzheimer's Disease. The methodcomprises administering a SMO of the invention, or a compositioncomprising a SMO of the invention, to a subject, wherein the SMOcontacts an OGA pre-mRNA and modulates the splicing of the OGA pre-mRNAto favor expression of naturally occurring splice variants which havereduced catalytic activity, as described elsewhere herein.

D. Method of Modulating Aph1B Receptor Pre-mRNA Splicing

In one embodiment, the present invention provides a method of modulatingsplicing of Aph1B pre-mRNA using a SMO to decrease expression orfunctionality of Aph1B in a subject. The method comprises administeringa SMO of the invention, or a pharmaceutical composition comprising a SMOof the invention, to a subject, wherein the SMO contacts an Aph1Bpre-mRNA and modulates the splicing of the Aph1B pre-mRNA to favorexpression of Aph1BΔ4, a variant in which exon 4 of Aph1B is deleted andis, thus, non-functional. Diseases and disorders where increasing Aph1Bexpression is believed to provide a therapeutic benefit to the subjectafflicted with the disease include, but are not limited to, Alzheimer'sDisease.

In another embodiment, the present invention provides a method oftreating a subject afflicted with Alzheimer's Disease. The methodcomprises administering a SMO of the invention, or a compositioncomprising a SMO of the invention, to a subject, wherein the SMOcontacts an Aph1B pre-mRNA and modulates the splicing of the Aph1Bpre-mRNA to favor expression of Aph1BΔ4, as described elsewhere herein.

E. Method of Modulating FOXM1 Receptor Pre-mRNA Splicing

In another embodiment, the present invention provides a method ofmodulating splicing of FOXM1 pre-mRNA using a SMO to decrease expressionor functionality of FOXM1 in a subject. The method comprisesadministering a SMO of the invention, or a composition comprising a SMOof the invention, to a subject, wherein the SMO contacts a FOXM1pre-mRNA and modulates the splicing of the Aph1B pre-mRNA to favorexpression of FOXM1Δ3 or FOXM1Δ6. Diseases and disorders whereincreasing FOXM1 expression is believed to provide a therapeutic benefitto the subject afflicted with the disease include, but are not limitedto, aberrant cell growth, cell differentiation, aberrant cell migration,tumorigenesis, or cancer including a liver cancer (The et al., 2002,Cancer Res. 62: 4773-80), a breast cancer (Wonsey et al., 2005, CancerRes. 65 (12): 5181-9), a lung cancer (Kim et al., 2006, Cancer Res. 66(4): 2153-61), a prostate cancer (Kalin et al., 2006, Cancer Res. 66(3): 1712-20; a cervical cancer of the uterus (Chan et al., 2008, J.Pathol. 215 (3): 245-52), a colon cancer (Douard et al., 2006, Surgery139 (5): 665-70), a pancreatic cancer (Wang et al., 2007, Cancer Res. 67(17): 8293-300), and a brain cancer (Liu et al., 2006, Cancer Res. 66(7): 3593-602).

In another embodiment, the present invention provides a method oftreating a subject afflicted with aberrant cell growth, celldifferentiation, aberrant cell migration, tumerigenesis, or cancer. Themethod comprises administering a SMO of the invention, or a compositioncomprising a SMO of the invention, to a subject, wherein the SMOcontacts a FOXM1 pre-mRNA and modulates the splicing of the FOXM1pre-mRNA to inhibit expression of FOXM1, as described elsewhere herein.

F. Method of Modulating HER3 Receptor Pre-mRNA Splicing

In one embodiment, the present invention provides a method of modulatingsplicing of HER3 pre-mRNA using a SMO to decrease expression orfunctionality of HER3 in a subject. The method comprises administering aSMO of the invention, or a composition comprising a SMO of theinvention, to a subject, wherein the SMO contacts a HER3 pre-mRNA andmodulates the splicing of the HER3 pre-mRNA. In one aspect, the SMOspecifically binds to the complementary sequence and enhances inclusionof intron 3 favoring expression of a truncated, non-functional HER3protein. In another aspect, the SMO specifically binds to thecomplementary sequence and enhances exclusion of exon 3 to favorexpression of HER3Δ3 to produce a non-functional protein. In stillanother aspect, the SMO contacts a HER3 pre-mRNA and enhances theexclusion of exon 11 to favor expression of HER3A 11 to produce anon-functional protein. Diseases and disorders where decreasing HER3expression is believed to provide a therapeutic benefit to the subjectafflicted with the disease include, but are not limited to, aberrantcell growth, cell differentiation, aberrant cell migration,tumerigenesis, cancer, and a metastatic cancer.

In another embodiment, the present invention provides a method oftreating a subject afflicted with aberrant cell growth, celldifferentiation, aberrant cell migration, tumerigenesis, or a cancer(Baselga et al., 2009, Nat Rev Cancer 9:463-475) including liver (The etal., 2002, Cancer Res. 62: 4773-80) breast, or a metastatic cancerderived from breast (Wonsey et al., 2005, Cancer Res. 65 (12): 5181-9),lung (Kim et al., 2006, Cancer Res. 66 (4): 2153-61), prostate (Kahn etal., 2006, Cancer Res. 66 (3): 1712-20; cervix of uterus (Chan et al.,2008, J. Pathol. 215 (3): 245-52), colon (Douard et al., 2006, Surgery139 (5): 665-70), pancreas (Wang et al., 2007, Cancer Res. 67 (17):8293-300), and brain (Liu et al., 2006, Cancer Res. 66 (7): 3593-602).The method comprises administering a SMO of the invention, or acomposition comprising a SMO of the invention, to a subject, wherein theSMO contacts a HER3 pre-mRNA and modulates the splicing of the Her3pre-mRNA to inhibit expression or function of HER3, as describedelsewhere herein.

G. Method of Modulating CypD Receptor Pre-mRNA Splicing

In one embodiment, the present invention provides a method of modulatingsplicing of CypD pre-mRNA using a SMO to inhibit expression orfunctionality of CypD in a subject. The method comprises administering aSMO of the invention, or a composition comprising a SMO of theinvention, to a subject, wherein the SMO contacts a CypD pre-mRNA andmodulates the splicing of the CypD pre-mRNA to favor expression ofCypDΔ1 or CypDΔ3 which exclude exons 1 and 3 respectively. Diseases anddisorders where decreasing CypD expression is believed to provide atherapeutic benefit to the subject afflicted with the disease include,but are not limited to, ALS, aberrant cell growth, cell differentiation,aberrant cell migration, tumerigenesis, Hepatitis B infection, and livercancer.

In another embodiment, the present invention provides a method oftreating a subject afflicted with amyotrophic lateral sclerosis (ALS;Sandyk, R., 2006, Int. J. Neurosci. 116: 775-826; Ionov, I. D., 2007,Amyotroph. Lateral Scler. 8:260-265). The method comprises administeringa SMO of the invention, or a composition comprising a SMO of theinvention, to a subject, wherein the SMO contacts a cyclophilin-Dpre-mRNA and modulates the splicing of the CypD pre-mRNA in the subject.

In another embodiment, the present invention provides a method oftreating a subject afflicted with aberrant cell growth, celldifferentiation, aberrant cell migration, tumerigenesis, hepatitis Binfection, and liver cancer. The method comprises administering a SMO ofthe invention, or a composition comprising a SMO of the invention, to asubject, wherein the SMO contacts a CypD pre-mRNA and modulates thesplicing of the CypD pre-mRNA to inhibit expression of CypD, asdescribed elsewhere herein.

Methods of Administration

Examples of methods for introducing oligonucleotides into cellsencompass in vivo and ex vivo methods. The oligonucleotides of theinvention, i.e. SMOs, are typically administered to a subject orgenerated in situ such that they hybridize with or bind to pre-mRNA of aspecific protein. In one embodiment, the pre-mRNA encodes a 5-HT2CR. Inanother embodiment, the SMO enhances inclusion of exon 5b duringsplicing of a 5-HT2CR pre-mRNA. In still another embodiment, thepre-mRNA encodes a glutamate receptor selected from the group consistingof GluR1-4. In yet another embodiment, the SMO modulates the ratio offlip and flop isoforms of any one of, or any combination of, the GluRs.In another embodiment, the pre-mRNA encodes OGA. In yet anotherembodiment, the pre-mRNA encodes Aph1B. In still another embodiment, thepre-mRNA encodes FOXM1. In still another embodiment, the pre-mRNAencodes HER3. In another embodiment, the pre-mRNA encodes CypD.

The hybridization can be by conventional Watson-Crick base pairing bynucleotide complementarity and/or wobble pairing of U-G or U-A nucleicacids to form a stable duplex. Hybridization can also occur, forexample, in the case of an oligonucleotide which binds to DNA duplexes,through specific interactions in the major groove of the double helix.

Conjugation of a SMO to a peptide, liposomes, colloidal polymericparticles as well as other means known in the art may be used to deliverthe oligonucleotides to a cell. The method of delivery selected willdepend at least on the cells to be treated and the location of the cellsand will be known to those skilled in the art. Localization can beachieved by liposomes, having specific markers on the surface fordirecting the liposome, by having injection directly into the tissuecontaining the target cells, by having depot associated in spatialproximity with the target cells, specific receptor mediated uptake, orthe like.

As described elsewhere herein and in the art, oligonucleotides may bedelivered using, e.g., methods involving liposome-mediated uptake, lipidconjugates, polylysine-mediated uptake, nanoparticle-mediated uptake,and receptor-mediated endocytosis, as well as additional non-endocyticmodes of delivery, such as microinjection, permeabilization (e.g.,streptolysin-O permeabilization, anionic peptide permeabilization),electroporation, and various non-invasive non-endocytic methods ofdelivery that are known in the art (refer to Dokka and Rojanasakul,Advanced Drug Delivery Reviews 44, 35-49, incorporated in its entiretyherein by reference). Methods of delivery may also include:

Cationic Lipids: Naked DNA can be introduced into cells in vivo bycomplexing the DNA with cationic lipids or encapsulating the DNA incationic liposomes. Examples of suitable cationic lipid formulationsinclude N-[-1-(2,3-dioleoyloxy)propyl]N,N,N-triethylammonium chloride(DOTMA) and a 1:1 molar ratio of1,2-dimyristyloxy-propyl-3-dimethylhydroxyethylammonium bromide (DMRIE)and dioleoyl phosphatidylethanolamine (DOPE) (see e.g., Logan, J. J. etal. (1995) Gene Therapy 2:38-49; San, H. et al. (1993) Human GeneTherapy 4:781-788).

Receptor-Mediated DNA Uptake: Naked DNA can also be introduced intocells in vivo by complexing the DNA to a cation, such as polylysine,which is coupled to a ligand for a cell-surface receptor (see forexample Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263:14621; Wilson etal. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No. 5,166,320).Binding of the DNA-ligand complex to the receptor facilitates uptake ofthe DNA by receptor-mediated endocytosis. A DNA-ligand complex linked toadenovirus capsids which naturally disrupt endosomes, thereby releasingmaterial into the cytoplasm can be used to avoid degradation of thecomplex by intracellular lysosomes (see for example Curiel et al. (1991)Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl.Acad. Sci. USA 90:2122-2126). Carrier mediated gene transfer may alsoinvolve the use of lipid-based compounds which are not liposomes. Forexample, lipofectins and cytofectins are lipid-based positive ions thatbind to negatively charged DNA and form a complex that can ferry the DNAacross a cell membrane. Another method of carrier mediated gene transferinvolves receptor-based endocytosis. In this method, a ligand (specificto a cell surface receptor) is made to form a complex with a gene ofinterest and then injected into the bloodstream. Target cells that havethe cell surface receptor will specifically bind the ligand andtransport the ligand-DNA complex into the cell.

Oligonucleotides may be directly introduced into the cell (i.e.,intracellularly); or introduced extracellularly into a cavity,interstitial space, into the circulation of an organism, introducedorally, or may be introduced by bathing a cell or organism in a solutioncontaining the RNA using methods known in the art for introducingnucleic acid (e.g., DNA) into cells in vivo. Vascular or extravascularcirculation, the blood or lymph system, and the cerebrospinal fluid aresites where the RNA may be introduced.

The oligonucleotides of the invention can be delivered to a subject byany art-recognized method. For example, peripheral blood injection ofthe oligonucleotides of the invention can be used to deliver thereagents via diffusive and/or active means. Alternatively, theoligonucleotides of the invention can be modified to promote crossing ofthe blood-brain-barrier (BBB) to achieve delivery of said reagents toneuronal cells of the central nervous system (CNS). Specific recentadvancements in oligonucleotide technology and delivery strategies havebroadened the scope of oligonucleotide usage for neuronal disorders(Forte, A., et al. 2005. Curr. Drug Targets 6:21-29; Jaeger, L. B., andW. A. Banks. 2005. Methods Mol. Med. 106:237-251; Vinogradov, S. V., etal. 2004. Bioconjug. Chem. 5:50-60; the preceding are incorporatedherein in their entirety by reference).

In certain embodiments, the oligonucleotides of the invention can bedelivered by transdermal methods (e.g., via incorporation of theoligonucleotide reagent(s) of the invention into, e.g., emulsions, withsuch oligonucleotides optionally packaged into liposomes). Suchtransdermal and emulsion/liposome-mediated methods of delivery aredescribed for delivery of antisense oligonucleotides in the art, e.g.,in U.S. Pat. No. 6,965,025, the contents of which are incorporated intheir entirety by reference herein.

The oligonucleotides of the invention may also be delivered via animplantable device (e.g., an infusion pump or other such implantabledevice). Design of such a device is an art-recognized process.

In one embodiment, a SMO is delivered directly into the cerebral spinalfluid (CSF) of a subject. Delivery of a SMO into the CSF of a subjectmay be accomplished by any means known in the art, including, but notlimited to, epidural injection or intrathecal injection via an infusionpump.

In one embodiment, SMOs are conjugated to a peptide to facilitatedelivery of the SMO across the blood brain barrier (BBB) followingparenteral administration to a subject. The SMO may be either directlyconjugated to the peptide or indirectly conjugated to the peptide via alinker molecule such as a poly amino acid linker, or by electrostaticinteraction. Peptides useful in delivering SMOs across the BBB include,but are not limited to, peptides derived from the rabies virusglycoprotein (RVG) that specifically bind to the nicotinic acetylcholinereceptor (AchR) present on neurons and the vascular endothelium of theBBB thereby allowing transvascular delivery, probably byreceptor-mediated transcytosis (Kumar et al., 2007, Nature 448:39-43,encompassed by reference in its entirety); Kunitz domain-derivedpeptides called angiopeps (Demeule et al., 2008, J. Neurochem.106:1534-1544; Demeule et al., 2008, J. Pharmacol. Exp. Ther.324:1064-1072).

Recombinant methods known in the art can also be used to achieveoligonucleotide reagent-induced modulation of splicing in a targetnucleic acid. For example, vectors containing oligonucleotides can beemployed to express, e.g., an antisense oligonucleotide to modulatesplicing of an exon of a targeted pre-mRNA.

For oligonucleotide reagent-mediated modulation of an RNA in a cell lineor whole organism, gene expression may be assayed by use of a reporteror drug resistance gene whose protein product is easily assayed. Suchreporter genes include acetohydroxyacid synthase (AHAS), alkalinephosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS),chloramphenicol acetyltransferase (CAT), green fluorescent protein(GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase(NOS), octopine synthase (OCS), and derivatives thereof. Multipleselectable markers are available that confer resistance to ampicillin,bleomycin, chloramphenicol, gentarnycin, hygromycin, kanamycin,lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.Depending on the assay, quantitation of the amount of gene expressionallows one to determine a degree of modulation which is greater than10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treatedaccording to the present invention. Lower doses of injected material andlonger times after administration of oligonucleotides may result inmodulation in a smaller fraction of cells (e.g., at least 10%, 20%, 50%,75%, 90%, or 95% of targeted cells). Quantitation of gene expression ina cell may show similar amounts of modulation at the level ofaccumulation of target mRNA or translation of target protein. As anexample, the efficiency of modulation may be determined by assessing theamount of gene product in the cell; pre-mRNA or mRNA may be detectedwith a hybridization probe having a nucleotide sequence outside theregion used for the oligonucleotide reagent, or translated polypeptidemay be detected with an antibody raised against the polypeptide sequenceof that region.

H. Method of Identifying SMOs for Skipping Exons

In general, SMOs function by sterically blocking or weakeninginteractions between elements of the spliceosomal complex and thepre-mRNA. Factors that influence whether an exon is spliced from itspre-mRNA and included in the mRNA include the strength of theintron-exon splice sites at either end of the exon, and on exonic andintronic regulatory motifs. In general, to facilitate exclusion(skipping) of exons from being included in mRNA of a targeted gene, theSMOs of the invention are designed to be complimentary to sequencesencompassing the 5′ and/or 3′ splice sites and/or ESEs and ISEs and arenot-complimentary to (avoid) ESSs and ISSs. Another major determinant ofthe functionality of SMOs are its thermodynamic properties. The skippingof exons from mRNA transcripts of targeted genes is enhanced by SMOs ofthe invention using the following set of methods.

(a) Ranking of 5′ Splice Site Strength

The relative strength of exonic 5′ splice sites is determined by thecombination of splice regulatory elements such as ESEs, ESSs, ISEs, andISSs, as well as how complementary the site is to the binding of the U1splicing factor. U1 splice site binding is ranked by two criterion: (i)complementarity (Roca, X. et al., 2005, RNA, 11: 683-698) and (ii)thermodynamics of U1 binding to the splice site (Garland, J. A. et al.,2004, Phys Rev E Stat Nonlin Soft Matter Phys, 69: 041903).

(b) Identification ESE/ESS/ISE Motifs

ESE motifs are defined using three prediction tools: ESE Finder(Cartegni, L. et al., 2003, Nucleic Acids Res, 31: 3568-3571),RESCUE-ESE (Fairbrother, W. G. et al., 2002, Science, 297: 1007-1013),and PESX (Zhang, X. H. et al., 2004, Genes Dev, 18: 1241-1250). ESSs aredefined using three prediction tools PESX, and a two hexamer data setanalysis by FAS-ESS (Wang, Z. et al., 2004, Cell, 119: 831-845).Finally, ISEs are predicted using the ACESCAN2 application (Yeo, G. W.et al., 2005, Proc Natl Acad Sci USA, 102: 2850-2855; Yeo, G. W. et al.,2007, PLoS Genet, 3: e85).

(c)RNA Structure and Oligo Walk

The Oligo Walk function of the publicly available “RNA Structure”program (Mathews, D. H. et al., 2004, Proc Natl Acad Sci USA, 101:7287-7292) is used to evaluate the predicted open secondary structure ofpre-mRNA sequences and the thermodynamic properties of the pre-mRNA.“RNA Structure” also provides analysis of thermodynamic parameters thatdetermine SMO binding strength and efficiency at a given site on thetarget pre-mRNA.

(1) Duplex ΔG^(o) ₃₇: Estimates the Gibbs free energy of the SMO topre-mRNA binding. More negative values for duplex ΔG^(o) ₃₇ will improveSMO binding to its target.

(2) Oligo-self ΔG^(o) ₃₇: Estimates the free energy of intramolecularSMO structures. More negative values indicate increasing stability ofintermolecular structures which may interfere with target binding.

(3) Oligo-oligo ΔG^(o) ₃₇: Provides the free energy of intermolecularSMO structures. Negative values indicate more stable SMO-SMO duplexes,thus values of oligo-oligo ΔG^(o) ₃₇ closer to zero will improve SMOfunctionality.

(4) T_(m): Estimates the melting temperature of SMO-target sequenceduplex formation. Higher T_(m) values will improve SMO binding andspecificity.

(5) Break-Target: Provides the energy penalty for breaking ofintramolecular RNA target base pairs when oligo is bound. Thus OptimalBreak-point ΔG^(o) ₃₇:≧0 kcal/mol

(d) BLAST Analysis of Potential Off-Target Hybridization

SMOs are screened using BLASTN analysis for potential hybridization tooff-target sites in the human genome. Generally, SMOs with greater than85% off-target hybridization to any other known pre-mRNA are eliminatedfrom consideration.

(e) Prioritization of SMOs Based on Combined Properties

SMOs are ranked for each of the five thermodynamic criterion withapproximate thresholds for criteria 1-3 as in (Matveeva, O. V. et al.,2003, Nucleic Acids Res, 31: 4989-4994) and criterion 4. Criterion 5 isranked but is not exclusionary. The thermodynamic criterion are combinedwith the information on splice site strength and splice enhancer motifsto establish candidate SMOs for empirical evaluation of splicingspecificity and efficiency.

It is apparent to someone skilled in the art that in most cases SMOs donot meet all criterion and there are necessary compromises made inselecting SMOs for empirical testing. For example a SMO and its targetpre-mRNA sequence may be exceptionally favorable from a thermodynamicstandpoint, and splice site strength and ESE elements may be strong.However, there may be predicted ESSs that would potentially lower SMOefficiency. The prioritization or weighting of the various factors inare taken into account on a case-by-case basis, when selecting SMOs at agiven gene target.

Certain SMOs of the invention are designed to skip ‘out of frame’ (OOF)exons (coding exon not divisible by 3) that are not alternativelyspliced. When constitutive OOF exons are skipped, the codon readingframe is shifted, resulting in an mRNA that encodes inappropriate aminoacids followed soon after by a pre-mature stop codon. The proteinproduced by such OOF exon skipping is non-functional and is degraded.This functions to block protein expression in a cell. Examples exonskipping of OOF exons for the purpose of preventing protein expressionin a cell are demonstrated elsewhere herein in the cases of FoxM1, HER3,and CypD.

IV. Pharmaceutical Compositions and Therapies

An SMO of the invention may be administered to a subject in apharmaceutical composition. As used herein the term “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions. Pharmaceutical compositions can be prepared as describedbelow.

Depending on the particular target RNA and the dose of oligonucleotidematerial delivered, this process may modulate function of the targetgene. In one embodiment of the instant invention, exon 5b-containing5-HT2CR protein production is enhanced in a treated cell, cell extract,organism or patient, with an enhancement of exon 5b-containing 5-HT2CRprotein levels of at least about 1.1-, 1.2-, 1.5-, 2-, 3-, 4-, 5-, 7-,10-, 20-, 100-fold and higher values being exemplary. In anotherembodiment of the invention, flop exon containing GluR proteinproduction is reduced in a treated cell, cell extract, organism, orpatient, with a decrease of flip exon GluR protein levels of at least1.1-, 1.2-, 1.5-, 2-, 3-, 4-, 5-, 7-, 10-, 20-, 100-fold and highervalues being exemplary. Enhancement of gene expression refers to thepresence (or observable increase) in the level of protein and/or mRNAproduct from a target RNA. Specificity refers to the ability to act onthe target RNA without manifest effects on other genes of the cell. Theconsequences of modulation of the target RNA can be confirmed byexamination of the outward properties of the cell or organism (aspresented below in the examples) or by biochemical techniques such asRNA solution hybridization, nuclease protection, Northern hybridization,reverse transcription, gene expression monitoring with a microarray,antibody binding, enzyme linked immunosorbent assay (ELISA), Westernblotting, radioimmunoassay (RIA), other immunoassays, and fluorescenceactivated cell analysis (FACS).

The oligonucleotide, i.e. the SMO, may be introduced in an amount whichallows delivery of at least one copy per cell. Higher doses (e.g., atleast 5, 10, 100, 500 or 1000 copies per cell) of material may yieldmore effective modulation; lower doses may also be useful for specificapplications.

Although the description of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as non-human primates, cattle, pigs, horses,sheep, cats, and dogs.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor ophthalmic, oral, parenteral, intranasal, buccal, or another routeof administration. Other contemplated formulations include projectednanoparticles, liposomal preparations, resealed erythrocytes containingthe active ingredient, and immunologically-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, intraocular,intravitreal, subcutaneous, intraperitoneal, intramuscular, intrasternalinjection, intratumoral, and kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e. powder or granular) form for reconstitution with asuitable vehicle (e.g. sterile pyrogen-free water) prior to parenteraladministration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may further comprise one or more of theadditional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, 0.1 to 20% (w/w)active ingredient, the balance comprising an orally dissolvable ordegradable composition and, optionally, one or more of the additionalingredients described herein. Alternately, formulations suitable forbuccal administration may comprise a powder or an aerosolized oratomized solution or suspension comprising the active ingredient. Suchpowdered, aerosolized, or aerosolized formulations, when dispersed,preferably have an average particle or droplet size in the range fromabout 0.1 to about 200 nanometers, and may further comprise one or moreof the additional ingredients described herein.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Remington's PharmaceuticalSciences (1985, Genaro, ed., Mack Publishing Co., Easton, Pa.), which isincorporated herein by reference.

The therapeutic and prophylactic methods of the invention thus encompassthe use of pharmaceutical compositions comprising a splice modifyingoligonucleotide of the invention to practice the methods of theinvention. The precise dosage administered will vary depending upon anynumber of factors, including but not limited to, the type of animal andtype of disease state being treated, the age of the animal and the routeof administration.

The compound may be administered to an animal as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even lessfrequently, such as once every several months or even once a year orless. The frequency of the dose will be readily apparent to the skilledartisan and will depend upon any number of factors, such as, but notlimited to, the type and severity of the disease being treated, the typeand age of the animal, etc. The formulations of the pharmaceuticalcompositions described herein may be prepared by any method known orhereafter developed in the art of pharmacology. In general, suchpreparatory methods include the step of bringing the active ingredientinto association with a carrier or one or more other accessoryingredients, and then, if necessary or desirable, shaping or packagingthe product into a desired single- or multi-dose unit.

III. Kits

Kits for practicing the methods of the invention are further provided.By “kit” is intended any manufacture (e.g., a package or a container)comprising at least one reagent, e.g., at least one SMO for specificallyenhancing inclusion of exon 5b in the 5-HT2C receptor for the treatmentof Prader-Willi Syndrome, a 5-HT2CR splicing deficit, hyperphagiaresulting from a 5-HT2CR splicing deficit, and/or symptoms ofobsessive-compulsive disorder resulting from a 5-HT2CR splicing deficit.In one embodiment, the kit includes at least one SMO directed to a GluRfor the treatment of epilepsy, a seizure disorder, or ALS. In stillanother embodiment, the kit includes at least one SMO directed to Aph1Bfor the treatment of Alzheimer's Disease. In yet another embodiment, thekit of the invention includes at least one SMO directed to OGA for thetreatment of Alzheimer's Disease. In a still further embodiment, the kitincludes at least one SMO directed to FOXM1 for the treatment of acarcinoma. In still another embodiment, the kit includes at least oneSMO directed to HER3 for the treatment of breast cancer. In yet anotherembodiment, the kit includes at least one SMO directed to CypD for thetreatment of ALS or liver cancer. The kit may be promoted, distributed,or sold as a unit for performing the methods of the present invention.Additionally, the kits may contain a package insert describing the kitand including instructional material for its use.

Positive, negative, and/or comparator controls may be included in thekits to validate the activity and correct usage of reagents employed inaccordance with the invention. Controls may include samples, such astissue sections, cells fixed on glass slides, etc., known to be eitherpositive or negative for the presence of the biomarker of interest. Thedesign and use of controls is standard and well within the routinecapabilities of those of ordinary skill in the art.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

The materials and methods employed in the experiments and the results ofthe experiments presented in these Examples are now described.

Experimental Example 1 Design and Validation of AntisenseOligonucleotides to Increase Inclusion of Exon 5b in the 5-HT2C Receptor

The snoRNA HBII-52 promotes inclusion of exon Vb in the 5-HT2C receptorby blocking a splice silencing element in the consensus region of5-HT2C. Thus an oligonucleotide identical to the consensus sequence ofMBII-52 (the mouse homolog) was designed to block the silencing site on5-HT2C. The first oligonucleotide was designed using a phosphorothioatelinkages between nucleotides and O-methyl substitutions on the 2′ riboseand is identical to the MBII-52 complementary box as follows:AUGCUCAAUAGGAUUACG (SEQ ID NO. 1).

Smaller SMOs may permit more specific targeting of inhibitory elementsin 5-HT2C. Therefore, a series of SMOs of varying lengths were designedusing an “antisense walk strategy” that has been recently used tosuccessfully target inhibitory regions in the SMN gene to correctsplicing (Hua et al., 2007, Public Library of Science Biol. 5:e73). TheSMO was “walked” base by base beginning with the 5′-most nucleotide (nt)aligned at the +3 nt position relative to the consensus sequence on5-HT2CR pre-mRNA and ending with the 3′-most nt of the oligomer at the−3 nt position of the consensus sequence. This strategy resulted in SMOsthat incrementally span the consensus region (Table 1 through Table 15).To ensure proper hybridization efficiency, these smaller SMOs may becomposed of an appropriate number of locked nucleic acid (LNA) residuessubstituted for 2′-O-methyl nucleotides.

To validate these SMOs, the SMOs are transfected into undifferentiatedNG108-15 cells that have previously been demonstrated to express both5-HT2CR splice isoforms (5a and 5b), the ratio of which can bedetectably altered by differentiation (Tohda et al., 2002, Jpn. J.Pharmacol. 90:138-144; Sukma et al., 2003, J. Pharmacol. Sci.92:433-436; Tohda et al., 2004, J. Pharmacol. Sci. 96:164-169). Cellsare harvested 48 hours post-transfection. Real-time PCR is used toquantify amounts of 5-HT2CR containing exon 5a and 5b. An SMO thatmimics the effect of MBII-52 increases the ratio of 5b to 5atranscripts. Western blotting is also performed using a rabbitpolyclonal antibody (Abcam) to quantify upregulation of full-length5-HT2CR protein.

Experimental Example 2 Phenotypic Improvement in Spinal Muscular Atrophy(SMA) Mice by SMO-Mediated Induction of SMN Expression

Spinal Muscular Atrophy (SMA) is caused by mutations in the SMN I gene,which encodes a protein called survival of motor neuron” or SMN, aubiquitous protein involved in RNA processing (Gubitz et al., 2004, Exp.Cell Res. 296:51-56; Monani, 2005, Neuron 48:885-896). The potential ofthe SMO developed by the Singh group (Singh et al., 2006, Mol. Cell.Biol. 26:1333-1346) to induce SMN expression and improve functionalperformance in vivo in the SMNΔ7^(+/+);SMN2^(+/+);Smn^(−/−) mice withsevere type 1 SMA phenotype was recently examined (Le et al., 2005, Hum.Mol. Genet. 14:845-857). First, to assess SMO distribution throughoutthe CNS, FAM-SMO was delivered intracerebroventricular (ICV) andfluorescent label was imaged in cryosections of brains and spinal cords(FIG. 1A-C). SMO was found to be broadly but not uniformly distributedthroughout the brain and spinal cord regions. These data were inaccordance with previous studies showing very effective CNSbiodistribution of SMOs of similar chemistry after both intrathecal andICV delivery (Smith et al., 2006, J. Clin. Invest. 116:2290-2296).

When SMA mice were given periodic intracerebroventricular injections ofSMO they showed greatly enhanced SMN expression at post-natal day 12 inboth brain and spinal cord, reaching 35-50% of the levels in wild-typelittermates (FIG. 1D-E). On average, SMN expression in the hippocampusregion of SMA mice injected with SMO was 34.4±1.8%, which wassignificantly greater than the 13.8±1.0% in un-injected SMA controls(2.5-fold increase; P<0.001). Importantly, the high level of SMNexpression in brain and spinal cord of SMA mice after ICV injection ofSMO alone was accompanied by a significant improvement in body weightduring post-natal development compared with un-treated SMA mice (FIG.1E). These data represent the highest level of SMN expression reportedto date in CNS of SMA mice. These data document that SMOs are broadlydistributed and biologically active in CNS after ICV delivery.

Experimental Example 3 Determination of the Ability of Optimal SMO toIncrease Inclusion of 5-HT2CR Vb In Vivo

SMOs are injected ICV into brains of normal mice (C57bl/6J) for 1 weekvia a cannula. Optimal dose and dosing regimens are around 2 μg/day for1 week, but can be optimized by the skilled artisan. Mouse brains areharvested one day after final injection, sub-regions are dissected out(hippocampus, cortex, hypothalamus) and trizol-extracted. Real time (RT)PCR is performed using primers previously shown to be able todistinguish Va and Vb splice variants (Kishore and Stamm, 2006, Science311:230-232). Since both splice variants are present and detectable innormal mouse brain (Canton et al., 1996, Mol. Pharm. 50:799-807),injection of SMOs will lead to a detectable increase in the ratio ofexon 5b to exon 5a-containing isoforms. Western blotting using a rabbitpolyclonal antibody (Abeam) is used to determine whether expression offull-length 5-HT2CR protein has been up-regulated.

Experimental Example 4 Examination of Functional Consequence ofModulating 5-HT2CR Splicing Using Electrophysiological Techniques inHippocampal Slices

By increasing inclusion of exon 5b in 5-HT2CR, the expression offunctional receptor is also increased. Both 5a and 5b-containingtranscripts are abundantly present in hippocampus (Canton et al., 1996,Mol. Pharmacol. 50:799-807), and it has been demonstrated elsewhereherein that SMO injected ICV can notably increase SMN levels in thisbrain region. Therefore initial electrophysiological assessment is inhippocampal slices. Activation of 5-HT2CR leads to an increase inintrinsic neuronal excitability and glutamate-mediated excitatorypostsynaptic current (EPSC) amplitudes in hippocampal CA1 pyramidalneurons (Beck, 1992, Synapse 10:334-340). These studies are done usingthe selective 5-HT2CR agonist Ro 60-175 (100 nM). The hippocampal slicepreparation and whole-cell patch clamping techniques are used to recordfrom CA1 pyramidal neurons. Intrinsic excitability is measured usingcurrent-clamp protocols to measure firing properties of neurons inresponse to voltage steps. EPSC measurements are done usingvoltage-clamp, and measuring synaptic responses to stimulation ofSchaffer collateral input. These techniques are described in (Tallentand Siggins, 1999, J. Neurophysiol. 81:1626-1635; Tallent et al., 2001,J. Neurosci. 21:6940-6948), incorporated herein by reference, in theirentirety. An enhancement in the ability of Ro 60-175 to increaseexcitability and EPSC amplitude after ICV injection of an optimal SMO isdue to increases in the expression of functional receptor in CA 1neurons.

Experimental Example 5 GluR Subunit Selection and SMO Design

The pre-mRNA splicing pattern of GluR subunits of the AMPA receptor isshown in FIG. 2. GluRs typically contain either of twomutually-exclusive alternative exons, flip or flop. Thus, the flip/flopexons constitute classical cassette exons, as opposed to constitutiveexons which are always retained in mRNA transcripts.

2′ OMe SMO that target exonic splice enhancers (ESEs) and splice site ofthe flip exons of GluRs are developed that facilitate specific skippingof flip exons of GluR pre-mRNAs by masking exon recognition by thespliceosome proteins. When an exon does not get spliced, it is removed(skipped) along with the introns on either side of it. The specific GluRflip subunits to be targeted as potential therapeutic agents fortreating ALS include GluR 3, GluR3+GluR4, GluR1, GluR1+GluR3, andGluR1+GluR2+GluR3+GluR4. Because of the nature ofconservation/divergence in ESEs and splice junctions of the flip exonsof GluRs, it is possible to selectively target any individual GluR forflip exon skipping, but it is not possible to target all possiblecombinations. For example it may be difficult to target ESEs of bothGluR1 and GluR3 in tandem without also impacting an ESE of GluR2. Itwould likely be even more difficult to target only the GluRs thatprovide Ca²⁺ permeability to the AMPA receptor (GluR1, GluR3, andGluR4), without also impacting GluR2. Given these constraints, GluRpre-mRNA targets for treating ALS would be as follows:

(a) Ranking of 5′ Splice Site Strength

The relative strength of exonic 5′ splice sites is determined by thecombination of splice regulatory elements such as ESEs, ESSs, ISEs, andISSs, as well as how complementary the site is to the binding of the U1splicing factor. U1 splice site binding is ranked by two criterion: (i)complementarity (Roca, X. et al., 2005, RNA, 11: 683-698) and (ii)thermodynamics of U1 binding to the splice site (Garland, J. A. et al.,2004, Phys Rev E Stat Nonlin Soft Matter Phys, 69: 041903).

(b) Identification ESE/ESS/ISE Motifs

ESE motifs are defined using three prediction tools: ESE Finder(Cartegni, L. et al., 2003, Nucleic Acids Res, 31: 3568-3571),RESCUE-ESE (Fairbrother, W. G. et al., 2002, Science, 297: 1007-1013),and PESX (Zhang, X. H. et al., 2004, Genes Dev, 18: 1241-1250). ESSs aredefined using three prediction tools PESX, and a two hexamer data setanalysis by FAS-ESS (Wang, Z. et al., 2004, Cell, 119: 831-845).Finally, ISEs are predicted using the ACESCAN2 application (Yeo, G. W.et al., 2005, Proc Natl Acad Sci USA, 102: 2850-2855; Yeo, G. W. et al.,2007, PLoS Genet, 3: e85).

(c)RNA Structure and Oligo Walk

The Oligo Walk function of the publicly available “RNA Structure”program (Mathews, D. H. et al., 2004, Proc Natl Acad Sci USA, 101:7287-7292) is used to evaluate the predicted open secondary structure ofpre-mRNA sequences and the thermodynamic properties of the pre-mRNA.“RNA Structure” also provides analysis of thermodynamic parameters thatdetermine SMO binding strength and efficiency at a given site on thetarget pre-mRNA.

(1) Duplex ΔG^(o) ₃₇: Estimates the Gibbs free energy of the SMO topre-mRNA binding. More negative values for duplex ΔG^(o) ₃₇ will improveSMO binding to its target.

(2) Oligo-self ΔG^(o) ₃₇: Estimates the free energy of intramolecularSMO structures. More negative values indicate increasing stability ofintermolecular structures which may interfere with target binding.

(3) Oligo-oligo ΔG^(o) ₃₇: Provides the free energy of intermolecularSMO structures. Negative values indicate more stable SMO-SMO duplexes,thus values of oligo-oligo ΔG^(o) ₃₇ closer to zero will improve SMOfunctionality.

(4) T_(m): Estimates the melting temperature of SMO-target sequenceduplex formation. Higher T_(m) values will improve SMO binding andspecificity.

(5) Break-Target: Provides the energy penalty for breaking ofintramolecular RNA target base pairs when oligo is bound. Thus OptimalBreak-point ΔG^(o) ₃₇: ≧0 kcal/mol

(d) BLAST Analysis of Potential Off-Target Hybridization

SMOs are screened using BLASTN analysis for potential hybridization tooff-target sites in the human genome. Generally, SMOs with greater than85% off-target hybridization to any other known pre-mRNA are eliminatedfrom consideration.

(e) Prioritization of SMOs Based on Combined Properties

SMOs are ranked for each of the five thermodynamic criterion withapproximate thresholds for criteria 1-3 as in (Matveeva, O. V. et al.,2003, Nucleic Acids Res, 31: 4989-4994) and criterion 4. Criterion 5 isranked but is not exclusionary. The thermodynamic criterion are combinedwith the information on splice site strength and splice enhancer motifsto establish candidate SMOs for empirical evaluation of splicingspecificity and efficiency.

Experimental Example 6 Measure Relative Efficacy of SMOs Using MouseLine Endogenously Expressing all Four GluR5

For analysis of SMO effectiveness, SMOs designed against the targetslisted in Table 2 through Table 7 are transfected into NSC-34 cellswhich are mouse neuroblastoma-spinal neuron hybrids that endogenouslyexpress all four mouse GluR5 (Eggett et al., 2000, J. Neurochem.74:1895-1902; Rembach et al., 2004, J. Neurosci. Res. 77:573-582). TheNSC-34 cell line is used widely as a culture model system for the studyof motor neurons (Cashman et al., 1992, Dev. Dyn. 194:209-221; Eggett etal., 2000, J. Neurochem. 74:1895-1902). NSC-34 cells were found toexpress low levels of GluR2 compared to GluR1, 3, and 4. This isconsistent with published reports that motor neurons are deficient inGluR2, thus rendering these cells vulnerable to calcium-mediated damageand excitotoxicity (Bar-Peled et al., 1999, Neuroreport 10:855-859;Heath et al., 2002, Neuroreport 13:1753-1757; Van et al., 2002, J.Neurophysiol. 88:1279-1287; Williams et al., 1997, Ann. Neurol.42:200-207). NSC-34 cells have also been shown to efficiently uptakeSMOs in culture (Rembach et al., 2004, J. Neurosci. Res. 77:573-582).Briefly, SMOs are complexed with lipofectamine and applied to NSC-34cells (100 SMO) in reduced serum medium for 4-6 hours (Cashman et al.,1992, Dev. Dyn. 194:209-221; Eggett et al., 2000, J. Neurochem.74:1895-1902). Medium is replaced and cells are grown for an additional24-48 hours in serum-containing medium and harvested. Cells are lysed,total RNA extracted (Trizol), and cDNA generated a reverse transcriptase(MultiScribe) using dNTPs and random hexamers. The level of both flip-and flop-containing mRNA transcripts is determined for each of the GluRsusing real-time PCR (TaqMan PCR system).

Next, SMOs that show the greatest decrease in the targeted flip isoformsare evaluated more extensively. The dose-response of lead SMOs areanalyzed by treating cells with concentrations ranging from 0-100 μM.Westerns blots are used to quantify GluR protein levels with antibodiesto GluR1 (1:100, AB5849; Chemicon), GluR2 (1:100, AB1768; Chemicon),GluR3 (1:1,500; (Gahring et al., 1998, Autoimmunity 28:243-248)), andGluR4 (1:100, AB1508; Chemicon). Toxicity is quantified by documentingmorphology of nuclei (DAPI), a known hallmark of cell damage.

An iterative process of SMO evaluation and optimization is used wherethe efficacy of the 2 top-ranked SMOs is performed, and these data usedto make the next SMO choices in a strategic manner. For example if a SMOshows a significant but incomplete reduction in flip isoform expression,bases are added or subtracted from either end to further improveefficacy.

Experimental Example 7 Determine Changes in ElectrophysiologicalProperties of AMPA Currents after Treatment with Lead SMOs

The SMOs that produce the most efficacious skipping of flip exons aretransfected into NSC-34 cells and AMPA-receptor mediated currents arestudied using whole cell patch clamp. Changes in flip/flop ratios ofGluRs change properties of AMPA receptor-mediated currents. Increases inthe flop to flip ratio result in the following changes in AMPA receptorcurrents: (i) An increase in desensitization kinetics (Sommer et al.,1990, Science 249:1580-1585). (ii) A decrease in the sensitivity tocyclothiazide (Johansen et al., 1995, Mol. Pharm. 48:946-955; Partin etal., 1994, Neuron 14:833-843) and an increase in the sensitivity to PEPA(Sekiguchi et al., 1998, Br. J. Pharmacol. 123:1294-1303). (iii) Adecrease in sensitivity for glutamate (Partin et al., 1995, Neuron14:833-843; Sommer et al., 1990, Science 249:1580-1585).

NSC-34 cells have also been shown to efficiently uptake SMOs in culture(Rembach et al., 2004, J. Neurosci. Res. 77:573-582). Briefly, SMOs arecomplexed with lipofectamine and applied to NSC-34 cells (100 μM SMO) inreduced serum medium for 4-6 hours (to induce differentiation (Eggett etal., 2000, J. Neurochem. 74:1895-1902; Rembach et al., 2004, J.Neurosci. Res. 77:573-582). Medium is replaced and cells grown for anadditional 24-48 hours in serum-containing medium and harvested. Cellsare lysed, total RNA extracted (Trizol), and cDNA generated with areverse transcriptase (MultiScribe) using dNTPs and random hexamers. Thelevel of both flip- and flop-containing mRNA transcripts is determinedusing real-time PCR using the TaqMan PCR system.

The whole-cell patch clamp method is used to record from treated anduntreated cells using a perfusion chamber. Cells are voltage-clamped at−70 mV and 1 mM or 10 mM glutamate or AMPA is applied using a rapidsuperfusion system. AP5 is used to block NMDA receptors. To evaluatedesensitization kinetics, 100 millisecond (ms) pulses of glutamate(Gardner et al., 2001, J. Neurosci. 21:7428-7437) are used.Desensitization kinetics are measured by fitting the decay of the AMPAcurrent with single and double exponentials using Clampfit software(Molecular Devices).

To determine cyclothiazide sensitivity, this drug (1-100 μM) isco-applied with 10 mM glutamate for 3 sec. For PEPA experiments, 10 mMglutamate and 1-1000 μM PEPA are co-applied for 1 sec. Dose-responsecurves are generated and desensitization kinetics determined asdescribed above. Difference in sensitivity to PEPA is greatest for GluR3flip vs. flop, so this drug may be especially useful in determining anincrease in GluR3 flop (Sekiguchi et al., 1998, Br. J. Pharmacol.123:1294-1303).

Glutamate sensitivity is determined by applying different concentrationsof glutamate (50 to 5000 μM) and generating dose-response curves ofmaximal current response. Since flip isoforms have a higher relativesensitivity to glutamate vs. kainate, the responsiveness of individualcells to 300 μM glutamate vs. 300 μM kainite is also assessed (Partin etal., 1995, Neuron 14:833-843; Sommer et al., 1990, Science249:1580-1585).

Experimental Example 8 In Vivo Application of SMOs

A cannula is implanted into the third ventricle (coordinates: midline,0.25 mm posterior to the bregma and 3 mm below the pial surface).Injection into the third ventricle (ICV) gives good access to thehippocampus (Chauhan et al., 2001, J. Neurosci. Res. 66:231-235). Fortyeight hours following surgery, delivery of SMO ICV is begun daily for 1week. SMOs are dissolved in sterile saline at 1 μg/μL. Optimal dose anddosing regimens can be determined by the skilled artisan, but based onprevious experience, is around 2 μg/day for 1 week. Mouse brains areharvested one day after final injection, hippocampus dissected out andtrizol-extracted. Real time (RT) PCR is performed using primerspreviously shown to specifically amplify flip and flop splice variants(Seifert et al., 2003, Mol. Cell. Neurosci. 22:248-258; Gomes et al.,2007, Mol. Cell. Neurosci. 37(2): 323-334). Significant changes insplicing are confirmed using Western blotting to determine if there aredetectable changes in GluR1 protein levels.

ICV injection of the SMOs (N=5) that target GluR3-flip and GluR1-flipwere made in neonatal FVB mice on postnatal days 1, 3, and 5. Controlinjections of saline were also made (N=4). ICV injection of the SMOsthat target GluR3-flip and GluR1-flip produce potent and specificreduction in targeted transcript expression in brain tissue harvested 24hours after the final administration of SMO (FIG. 3). Flip and floptranscript levels of all GluRs were measured using real-time PCR. Boththe GluR3 and the GluR1SMOs produced nearly complete reduction intargeted transcription expression with no significant effect on otherGluR isoforms. Decreasing flip in principle neurons and glia isprotective against seizures (Seifert et al., 2004, J. Neurosci.24:1996-2003; Ge et al., 2006, Science 312:1533-1537).

ICV injections of the SMOs that target all four GluR flip isoformsneonatal FVB mice on postnatal days 1, 3, and 5 produce potent reductionin GluR1, GluR2, and GluR3 flip transcript expression in brain tissueharvested 24 hours after the final administration of SMO (FIG. 4). Aconcomitant increase in flop transcripts was also observed.

Experimental Example 9 Efficacy of SMO in Modulating Seizure Activity inMice

Neonatal mice were administered ICV injections of GluR1SMO on postnatalday 1, 3, and 5 and tested for seizure activity on postnatal day 10.Control ICV injections of saline were also done. Seizures were inducedvia an intraperitoneal injection of kainic acid and the stage of seizurewas evaluate from the least severe (stage 3) to status epilepticus(stage 6). GluR1SMO administration significantly reduces the percent ofmice entering stage 4, stage 5, and stage 6 seizures (FIG. 5).

Experimental Example 10 Using SMOs to Target HER3 and Treat BreastCancer

SMOs as described elsewhere herein are developed which potently andspecifically reduce HER3 expression in a cell, reduce tumorigenesis ofHER2 overexpressing breast cancer cells (HOBCs) in vitro, and blockmetastasis in vivo. The SMOs specifically modulate HER3 pre-mRNAsplicing, resulting in downregulation of functional full-length HER3.All SMOs are synthesized using of 2′MOE chemistry and designed to targetidentified naturally occurring non-functional alternative splicevariants of HER3, as well as novel isoforms. HER3-specific SMOs areevaluated for efficacy by transfecting HOBC lines (including SKBR3,BT474, and MDA-MB-453 cell lines). Changes in HER3 expression in cellstransfected with SMOs are evaluated using real-time PCR and Western blotanalysis to determine the level of HER3 expression at the nucleic acidand protein level. The effects of SMOs on activation of Akt pathway inbreast cancer cells using phosphospecific antibodies is also done. Celllines are also transfected with scrambled SMOs as a negative controls.

SMOs are evaluated in HBOCs (primarily SKBR3 cells) by measuring severalindices of oncogenic activity including effects on: (i) growth in softagar, (ii) survival from matrix detachment, and (iii) invasion usingtranswell invasion assays.

Liver is a primary site of metastasis of HOBCs. SMOs localize mostspecifically to liver after IV and IP delivery (Yu et al., 2009,Biochem. Pharmacol. 77:910-919). The efficacy of SMOs directed againstHER3 in blocking breast cancer cell metastasis in liver is evaluated asfollows. SKBR3 cells (1×10⁶), stably transformed to express luciferasereporter, are administered through the tail vein of scid mice (N=10),immediately followed by IV injection of an HER3 targeted SMO. SMOs areinjected weekly (IV) for about 6 weeks. The determination of the optimalinterval for administering a SMO is well without routine experimentaloptimization in the art. Metastasis in liver and other organs isvisualized with the quantitative IVIS Lumina Imaging System. Livers arethen removed and analyzed for indices of macro and micrometastasis. HER3expression is measured using immunohistochemistry. Mice (N=10) injectedwith SKBR3 cells and scrambled SMOs are controls.

Experimental Example 11 Using SMOs to Target OGA to Reduce TauHyperphosphorylation in Treat Alzheimer's Disease

SMOs which target splicing of both human and mouse OGA pre-mRNA togenerate splice isoforms with dominant negative properties and reducedcatalytic efficiency have been developed according to the methodsdescribed elsewhere herein. Exemplary SMOs targeted to produce theOGA10t and OGAΔ8 isoforms are depicted in Table 7 and Table 8.

SMOs are evaluated for their effect on O-GlcNAc levels by western blotof total protein in a cell using anti-O-GlcNAc antibody CTD110.6(Dorfmueller et al., 2009, Biochem. J. 420:221-227). OGA splice isoformswith lowered catalytic activity result in increases of O-GlcNAcylationof proteins, since OGA will continue to attach O-GlcNAc residues onnuclear and cytoplasmic targets more rapidly than they can be removed(Yuzwa et al., 2008, Nat. Chem. Biol. 4:483-490).

SMOs are specifically delivered to the CNS by ICV injection to avoid offtarget peripheral effects. SMOs are delivered using short termcontinuous infusion of a pharmaceutical composition comprising an SMO bya stereotaxically implanted cannulae in the lateral brain ventricle andconnected to a sub-cutaneously implanted osmotic pump (Alzet). Normalmice are administered either saline or a dose of SMO ranging from 1-10μg of SMO daily for 3 weeks (Smith et al., 2006, J. Clin. Invest.116:2290-2296). During the 3 weeks of SMO infusion, mice are evaluatedweekly for declarative and spatial memory, and motor deficits by Morriswater maze, novel object recognition, and rotarod testing.

Following the period of SMO administration, mice are euthanized andbrain tissue (including cortex and hippocampus) extracted for testing.Real-time PCR performed on brain sections to determine transcript levelsof the desired OGA10t or OGAΔ8 alternative splice isoforms. Brain tissuefrom saline and SMO dosed mice is also be evaluated by western blot forglobal increases in O-GlcNAc levels.

Triple Transgenic Alzheimer's (3× Tg) mice are administered SMOs at adose which provides optimal effects on increasing O-GlcNAc levels. The3× Tg mice are transgenic for PS1_(M146V), APP_(Swe), and tau_(p301L)mutations and demonstrate earlier onset of cognitive and synapticdysfunction as compared to other AD mouse models. Onset of obviouspathology in 3×-Tg mice occurs at 6 months of age with the presence ofsynaptic and cognitive deficits and at 12 months the presence of tauimmunoreactivity can be detected (Oddo et al., 2003, Neuron 39:409-421;Pietropaolo et al., 2008, Behav. Neurosci. 122:733-747). Thus, SMOtreatment from 11-12 months of age when tau should be in ahyperphosphorylated state in addition to the presence of synaptic andcognitive deficits due to Aβ deposition, allows for short termevaluation of the effects of increased O-GlcNAc levels on overallcognitive symptoms as well as tau phosphorylation state.

Eleven month old 3× Tg mice are treated with saline or an SMO using theICV infusion method described elsewhere for 3 weeks. During infusionperiod, mice are evaluated weekly for cognitive, memory, and motordeficits by Morris water maze, novel object recognition, and rotarodtesting. These mice are also tested for effect on total brain O-GlcNAclevels. The mice are euthanized after 3 weeks of infusion (at ˜12 monthsof age). Brain tissue samples from SMO treated 3× Tg mice is evaluatedat the end of the dosing period for total O-GlcNAcylation levels bywestern blot as compared to saline controls.

The effect of SMO that target OGA pre-mRNA on tau phosphorylation isevaluated using the same protocol described above. Samples are takenfrom the cortex and hippocampus and evaluated for total tauphosphorylation using tau epitope 5 antibody, modification-statespecific antibodies (pSer422, pSer262, pSer396, and pThr231), and tauepitope 1 antibody directed against non-phosphorylated residues atSer198, Ser199 and Ser202. By using this panel of antibodies, changes inphosphorylation state of all the relevant phosphorylation sites isevaluated by Western blot (Yuzwa et al., 2008, Nat. Chem. Biol.4:483-490). Prevention of tau phosphorylation at these residues byaltering splicing of OGA pre-mRNA will block progression of taupathology in AD.

Experimental Example 12 Using SMOs to target Aph1B to Treat Alzheimer'sDisease

A “triple-transgenic” mouse model, 3×-tg AD mice, expresses mutant APP,PSN1 (presenilin), and tau transgenes. These mice have cognitive andsynaptic dysfunction similar to those in other AD mice, but with earlieronset (Oddo et al., 2003, Neuron 39:409-421). Specifically, the 3×-tg Admice show significant memory deficits when tested using the Morris WaterMaze paradigm as early as 120 days of age.

SMOs that target Aph1B, as exemplified by oligonucleotides listed inTable 9, are used to modulate splicing Aph1B pre-mRNA. An SMO thattargets Aph1B pre-mRNA is infused ICV for about a 3 week periodbeginning at 100 days of age. This provides adequate time for the SMO toexert its effect on Aph1B pre-mRNA splicing. In addition, mice are ˜4months of age at the end of the infusion period when they are evaluatedfor changes in cognitive performance.

For continuous delivery of SMO to the CSF, mice are cannulatedstereotaxically into the lateral ventricle, with the cannula tubingalready connected to a sub-cutaneously implanted Alzet mini pumppre-loaded with a pharmaceutical composition comprising a SMO. Thepharmaceutical composition comprising the SMO is equilibrated for 2 daysin sterile saline at 37° C. In this system, the cannulae, tubing, andpump is surgically implanted beneath the skin. The model pump used inthese experiments delivers its contents at a constant rate of 4 μL perday and holds enough volume (100 μL) to last about 25 days. We usedosing rates of 1 to 10 μg SMO per day in mice.

Examples of SMOs that specifically skip exon 4 of the Aph1B pre-mRNA areprovided in Table 9. These SMOs were developed according to thefollowing rational: Aph1B naturally expresses a non-functionalalternative splice variant missing exon 4 (Saito et al., 2005, Biochem.Biophys. Res. Comm. 330:1068-1072). Alternatively spliced exons areknown to be more readily modulated by oligomers than constitutive exons.Second, exon 4 of Aph1B contains a conserved GXXXG motif, critical forthe assembly and activity of the γ-secretase complex (Lee et al., J.Biol. Chem. 279:4144-4152). Thus, Aph1B protein missing exon 4 isnon-functional and unstable (Saito et al., 2005, Biochem. Biophys. Res.Comm. 330:1068-1072).

An SMO is transfected into neuroblastoma SY5Y cells that expressγ-secretase. SMOs are complexed with lipofectamine and applied to SY5Ycells at a concentration of 100 μM SMO for 4 hours. The bathing mediumis replaced and cells are maintained in culture for an additional 24hours before they are and harvested. RNA is extracted using standardtechniques known in the art, and cDNA generated with superscript RTusing oligo-dT and random hexamers. The level of Aph1B mRNA transcriptswith and without exon 4 is determined using Real-time PCR. Thedose-response of lead SMOs will is analyzed using Westerns blots toquantify Aph1B protein expression in transfected cells. Toxicity is alsoquantified by documenting morphology of nuclei and using DAPI staining,a known hallmark of cell damage (Martin et al., 2005, Cytometry Part A67A:45-52).

At the end of the infusion period, mice are evaluated for changes incognitive performance using the Morris Water Maze test with trainingbeginning at the end of the 3 week drug infusion period. The MorrisWater Maze comprises a 25 gallon tub 71 cm in diameter and 33 cm highcontaining water maintained at 23° C. A platform 6 cm in diameter isplaced in the center of one quadrant. The mice have 2 blocks of 4visible platform trials where they are given 60 seconds to reach theplatform, with a 5 min inter-trial interval (Varvel et al., 2005,Psychopharmacol. (Berl) 179:863-872). Location of the platform ischanged semi-randomly between trials. Mice are videotaped and latency toreach the platform is recorded. The following day the mice begin hiddenplatform training where the platform is submerged in opaque water. 2blocks of 4 trials each (5 minute inter-trial interval) are run each dayfor 4 days, with 1 hour between blocks. A trial consists ofsemi-randomly placing the mouse in one of three quadrants without theplatform and giving the mouse 60 seconds to locate the platform. Theplatform remains in the same location for the 4 days of hidden platformtraining.

Probe tests are run 24 and 72 hours after the final day of training. Theplatform is removed from the tub and the mice are semirandomly placed inone of the four quadrants and allowed to swim for 30 seconds. After the24 hour probe the platform is placed back in the same location and themouse allowed to find it, to minimize extinction. The percent time spentin each quadrant is recorded. A one way ANOVA is run (Statistica) todetermine significance in probe trials. A repeated measures ANOVA isused to determine differences in latencies to reach the platform duringtraining, with a post-hoc Tukey's test to determine where thesignificant differences occur.

Following the final behavioral test, mice are euthanized and brainsrapidly removed. The left and right hippocampus and cortex are quicklyexcised. To alleviate biases due to potential differences between theleft and right hemisphere tissues, each assay is performed on an equalnumber of left and right hemisphere tissues. RNA is immediatelyextracted from half of the hippocampus and cortex tissues, and cDNAprepared according to standard techniques known in the art. Theremaining tissue is immediately processed for protein extraction and thepreparation of a soluble fraction and a membrane fraction. Both solubleand membrane fraction preparations will be treated as described below.

Transcript and protein levels of Aph1B in the hippocampus and cortex aremeasured for all groups. Transcript levels of Aph1B will be measured byReal-time PCR, using custom primer-probe sets using GAPDH as theinternal control. Aph1B protein content is measured by Western blotanalysis of the soluble and membrane fractions with a polyclonalantibody (Santa Cruz; sc-49358).

Aβ40 and Aβ42 levels in hippocampus and cortex are measured in both thesoluble and membrane fractions using sandwich ELISA with antibodiesagainst human Aβ40 (2G3antibody) and human Aβ42 (21F12 antibody), bothdetected with biotin-3D6 antibody (Kanekivo et al., 2009, J. Biol. Chem.284:33352-33359)

In addition, extracts from the various brain sections are also be probedfor changes in activated Notch intracellular domain (NICD), the welldocumented released product of Notch cleavage by γ-secretase. Anotherknown non-amyloidal substrate of γ-secretase is N-cadherin. Western blotanalysis will be used to measure levels of NICD (Cell Signaling) andN-cadherin (Santa Cruz; sc-7939).

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1-54. (canceled)
 55. A composition comprising a splice modulatingoligonucleotide (SMO) that specifically binds a complementary sequenceof a pre-mRNA that undergoes splicing to form a mRNA encoding a proteinselected from a glutamate activatedα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptorsubunit (GluR), 5-HT2CR, HER3, Aph1B, CypD, OGA, or FOXM1. 56-61.(canceled)
 62. The composition according to claim 55, wherein said SMOis selected from any of SEQ ID NOs: 1-1089, or a sequence having up to90% identity with any of SEQ ID NOs: 1-1089.
 63. The compositionaccording to claim 55, wherein at least one nucleotide in said SMOcontains a non-naturally occurring modification comprising at least oneof a chemical composition of phosphorothioate 2′-O-methyl,phosphorothioate 2′-MOE, locked nucleic acid (LNA), peptide nucleic acid(PNA), phosphorodiamidate morpholino, or any combination thereof. 64.The composition according to claim 55, further comprising apharmaceutically acceptable carrier.
 65. A method of treating a disease,disorder or condition selected from hyperexcitability, excitotoxicity,Prader-Willi Syndrome, hyperphagia, hyperphagia induced obesity,Angelman Syndrome, obsessive/compulsive disorder, depression, psychoticdepression, major depressive disorder, bipolar disorder, sleepimpairment, autism, schizophrenia, Parkinson's disease, drug addiction,spinal cord injury, traumatic brain injury, neuropathic pain, diabetes,amyotrophic lateral sclerosis (ALS), epilepsy, Alzheimer's disease,hepatitis B virus (HBV) infection, hepatocellular carcinoma, breastcancer, liver cancer, lung cancer, prostate cancer, cervical cancer,colon cancer, pancreatic cancer, or brain cancer, in a subject in needthereof, comprising administering a composition according to claim 55.66. A composition comprising a splice modulating oligonucleotide (SMO)that specifically binds a complementary sequence of a pre-mRNA thatundergoes splicing to form a mRNA encoding a glutamate activated AMPAreceptor subunit (GluR).
 67. The composition according to claim 66,wherein said GluR is selected from GluR1, GluR2, GluR3, or GluR4, or anycombination thereof.
 68. The composition according to claim 66, whereinsaid SMO is selected from any of SEQ ID NOs: 57-526, or a sequencehaving up to 90% identity with any of SEQ ID NOs: 57-526.
 69. Thecomposition according to claim 68, wherein said SMO is selected from anyof SEQ ID NOs: 228, 123, 243, 269, 306, 318, 442, or 496, or a sequencehaving up to 90% identity with any of SEQ ID NOs: 228, 123, 243, 269,306, 318, 442, or
 496. 70. The composition according to claim 66,wherein at least one nucleotide in said SMO contains a non-naturallyoccurring modification comprising at least one of a chemical compositionof phosphorothioate 2′-O-methyl, phosphorothioate 2′-MOE, locked nucleicacid (LNA), peptide nucleic acid (PNA), phosphorodiamidate morpholino,or any combination thereof.
 71. The composition according to claim 66,further comprising a pharmaceutically acceptable carrier.
 72. A methodof treating a disease, disorder or condition associated withhyperexcitability or excitotoxicity in a subject in need thereof,comprising administering a composition according to claim
 66. 73. Themethod according to claim 72, wherein the SMO in the compositionspecifically binds to a complementary sequence on a pre-mRNA in at leastone of the group consisting of an intron-exon splice site, an exonicsplice enhancer (ESE) site, and an intronic splice enhancer (ISE) site,thereby excluding a selected exon from the mRNA encoding the GluR, andaltering expression of a GluR or one or more isoforms thereof.
 74. Themethod according to claim 73, wherein the selected exon is a GluR-flipexon.
 75. The method according to claim 72, wherein said administrationdecreases hyperexcitability or excitotoxicity, increases AMPA channeldesensitization, decreases glutamate sensitivity of AMPA channels, ordecreases calcium ion conductance through AMPA channels.
 76. A method oftreating a disease, disorder or condition associated withhyperexcitability or excitotoxicity in a subject in need thereof,comprising administering a composition according to claim
 66. 77. Amethod of treating amyotrophic lateral sclerosis (ALS), epilepsy, ortraumatic brain injury, comprising administering a composition accordingto claim 66.